Repeat dose oral toxicity of 4-methylbenzoic acid was studied in rats according to the guideline for the 28-day repeated dose toxicity test in mammalian species (Chemical substances control law of Japan). No deaths were observed in any treatment group. In the 1000 mg/kg-treated group, temporary salivation was observed in a small number of male and female rats. Increase in food consumption became apparent from 8 treatments in the 1000 mg/kg-treated group. Hematological and biochemical analyses at the terminal killing revealed that the treatment with 1000 mg/kg decreased platelet counts and increased AST activity in the females. No changes were observed in clinical signs, on detailed clinical observation, or for pathological findings. The NOEL is considered to be 300 mg/kg/day for males and females.
A preliminary reproduction toxicity screening test of 4-methylbenzoic acid was conducted in rats according to the OECD Test Guideline 421. 4-Methylbenzoic acid at a dose of 1000 mg/kg caused salivation in both sexes of animals, a reduction in absolute and relative epididymal weights in males and a slight increase in food consumption in females. No other effects of the compound concerning general toxicity were found in any group. While no effect of the compound was observed on estrous cycle, mating performance, ovulation and lactation at any doses, 300 mg/kg and more of the compound increased pre-implantation loss of embryos. In addition, 1000 mg/kg of the compound increased the number of epidydimal lumina with reduction in the number of sperm. The NOEL for general toxicity of 4-methylbenzoic acid is considered to be 300 mg/kg/day in both sexes of animals. The NOEL for reproductive/developmental toxicity is considered to be 100 mg/kg/day in both sexes of animals, and that for the offspring to be 1000 mg/kg/day.
4-Methylbenzoic acid was not mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 or Escherichia coli WP2 uvrA/pKM101, with or without an exogenous metabolic activation system.
4-Methylbenzoic acid induced structural chromosomal aberrations in CHL/IU cells without exogenous metabolic activation system. Polyploidy was not induced in any treatment group.
| Purity | : | 98.85 % |
| Test species/strain | : | Rat/Crj:CD(SD)IGS |
| Test method | : | OECD Test Guideline 401 |
| Route | : | Oral (Gavage) |
| Dosage | : | 0 (Vehicle), 1000, 1500, 2000 mg/kg |
| Number of animals/group | : | Males, 5; females, 5 |
| Vehicle | : | 0.5 % Sodium carboxymethylcellulose solution |
| GLP | : | Yes |
Test results:
Reduction in body weight gain was found in females given 1500 mg/kg and more. However, body weight gain during the observation days 4-8 was higher than that in the controls. In the males, no effects were evident on body weight increase.
No abnormalities were found at necropsy of any of the animals.
| Purity | : | 98.85 % |
| Test species/strain | : | Rat/Crj:CD(SD)IGS |
| Test method | : | Guideline for the 28-Day Repeated Dose Toxicity Test in Mammalian Species (Chemical Substances Control Law of Japan) |
| Route | : | Oral (Gavage) |
| Dosage | : | 0 (Vehicle), 100, 300, 1000 mg/kg |
| Number of animals/group | : | Males, 10; females, 10 (0 and 1000 mg/kg) Males, 5; females, 5 (100 and 300 mg/kg) |
| Vehicle | : | 0.5 % Sodium carboxymethylcellulose solution |
| Administration period | : | Males and females, 28 days |
| Terminal killing | : | Males and females, on days 29 and 43 |
| GLP | : | Yes |
Test results:
From the above results, the NOEL is considered to be 300 mg/kg/day for males and females.
| Purity | : | 98.85 % |
| Test species/strain | : | Rat/Crj:CD(SD)IGS |
| Test method | : | OECD Test Guideline 421 |
| Route | : | Oral (Gavage) |
| Dosage | : | 0 (Vehicle), 100, 300, 1000 mg/kg |
| Number of animals/group | : | Males, 13; females, 13 |
| Vehicle | : | 0.5 % Sodium carboxymethylcellulose solution |
| Administration period | : | Males, 42 days Females, from 14 days before mating to day 3 of lactation |
| Terminal killing | : | Males, day 43 Females, day 4 of lactation Offspring, 4 days after birth |
| GLP | : | Yes |
Test results:
Neither deaths nor a moribund condition were observed in any animals. Except for salivation during the later period of the treatment in the 1000 mg/kg-treated group, the compound did not cause any clinical signs.
The compound did not affect male body weight gain or food consumption, although it reduced absolute and relative epidydimal weights at the dose of 1000 mg/kg. Testicular weights were comparable among the groups.
Reproductive toxicity was noted for the compound, such as decreases in the implantation index and the number of offspring, reduced body weight gain of females during late pregnancy and lactation in the 300 mg/kg and more-treated groups. As for food consumption, a slight increase was observed at the beginning of the treatment and on gestational days 14-15 in the 1000 mg/kg-treated group. During the lactation period, physiological changes related to the reduction in number of pups caused a decrease in food consumption. The compound did not cause abnormal findings in any females evident at the necropsy.
<Reproductive/developmental toxicity>
No abnormalities were found in the testis at the histopathological examination. However treatment with 1000 mg/kg increased the number of cauda epidydimal lumina with reduction in the number of spermatozoa and slightly increased cell debris.
In the females, treatment with the compound up to 1000 mg/kg did not alter the estrous cycle and the number of corpora lutea. Indices for mating performance, such as the copulation index, the incidence of vaginal estrus and pairing days until copulation, were not affected by the treatment at any dose level. Fertility of the 1000 mg/kg-treated group, however, was reduced to 60 %, and the implantation index and the number of newborn were reduced in the groups treated with 300 mg/kg and more. Reduction in the number of newborn in the 1000 mg/kg-treated group was accompanied by reduction in the number of live newborn and live pups on day 4 of lactation.
No abnormalities were observed in delivery and lactation conditions, and the treatment did not alter gestation length. The treatment did not affect the indices of birth, live birth and viability on day 4 of lactation. In addition, the compound did not affect indices for development and growth of the offspring, such as sex ratios and body weights on days 0 and 4 of lactation and morphology, at any dose level.
<Evaluation>
From the above results, the NOEL for general toxicity of 4-methylbenzoic acid is considered to be 300 mg/kg/day in both sexes of animals. The NOEL for reproductive/developmental toxicity is considered to be 100 mg/kg/day in both sexes of animals, and the NOEL for the offspring to be 1000 mg/kg/day.
| Purity | : | 98.85 % |
| Test species/strain | : | Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA/pKM101 |
| Test method | : | Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan) and OECD Test Guideline 471 |
| Procedures | : | Pre-incubation method |
| Solvent | : | Dimethyl sulfoxide |
| Positive controls | : | -S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (TA100, TA98), Sodium azide (TA1535), 9-Aminoacridine hydrochloride (TA1537) and N-Ethyl-N'-nitro-N-nitro-soguanidine (WP2 uvrA/pKM101) +S9 mix; 2-Aminoanthracene (all strains) |
| Dosage | : | -S9 mix; 0, 313, 625, 1250, 2500, 5000 μg/plate (all strains) +S9 mix; 0, 156, 313, 625, 1250, 2500, 5000 μg/plate (all strains) |
| S9 | : | Rat liver, induced with phenobarbital and 5,6-benzoflavone |
| Plates/test | : | 3 |
| Number of replicates | : | 2 |
| GLP | : | Yes |
Test results:
Genetic effects:
Salmonella typhimurium TA100, TA1535, TA98, TA1537
| + | ? | - | |
| Without metabolic activation: | [ ] | [ ] | [*] |
| With metabolic activation: | [ ] | [ ] | [*] |
Escherichia coli WP2 uvrA/pKM101
| + | ? | - | |
| Without metabolic activation: | [ ] | [ ] | [*] |
| With metabolic activation: | [ ] | [ ] | [*] |
| Purity | : | 98.85 % |
| Type of cell used | : | Chinese hamster CHL/IU cells |
| Test method | : | Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan) and OECD Test Guideline 473 |
| Solvent | : | 1 % Sodium carboxymethylcellulose solution |
| Positive controls | : | -S9 mix; Mitomycin C +S9 mix; Benzo[a]pyrene |
| Dosage | : | -S9 mix(6 hr short-term treatment)[Main test, without buffering of the medium]; 0, 500, 1000, 1500, 2000 μg/mL -S9 mix(6 hr short-term treatment)[Confirmation test 1, without buffering of the medium]; 0, 1400, 1600, 1800, 2000 μg/mL -S9 mix(6 hr short-term treatment)[Confirmation test 1, with buffering of the medium]; 0, 1400, 1600, 1800, 2000 μg/mL -S9 mix(6 hr short-term treatment)[Confirmation test 2, with buffering of the medium]; 0, 1250, 2500, 5000 μg/mL +S9 mix(6 hr short-term treatment)[Main test, without buffering of the medium]; 0, 250, 500, 1000, 1500 μg/mL +S9 mix(6 hr short-term treatment)[Confirmation test 1, without buffering of the medium]; 0, 1000, 1200, 1400 μg/mL +S9 mix(6 hr short-term treatment)[Confirmation test 1, with buffering of the medium]; 0, 1000, 1200, 1400 μg/mL +S9 mix(6 hr short-term treatment)[Confirmation test 2, with buffering of the medium]; 0, 1250, 2500, 5000 μg/mL -S9 mix(24 hr continuous treatment)[Main test, without buffering of the medium]; 0, 250, 500, 1000, 1500 μg/mL -S9 mix(24 hr continuous treatment)[Main test, with buffering of the medium]; 0, 1250, 2500, 5000 μg/mL -S9 mix(24 hr continuous treatment)[Confirmation test, with buffering of the medium]; 0, 1000, 2000, 3000 μg/mL |
| S9 | : | Rat liver, induced with phenobarbital and 5,6-benzoflavone |
| Plates/test | : | 2 |
| GLP | : | Yes |
Test results:
| Lowest concentration producing cytogenetic effects in vitro | : | ||
| Without metabolic activation (continuous treatment) | : | 100 μg/mL (clastogenicity) | |
Genetic effects:
| clastogenicity | polyploidy | |||||
| + | ? | - | + | ? | - | |
| Without metabolic activation: | [*] | [ ] | [ ] | [ ] | [ ] | [*] |
| With metabolic activation: | [ ] | [ ] | [*] | [ ] | [ ] | [*] |
| 1) | The tests were performed by the Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa, 257-8523, Japan. Tel +81-463-82-4751, Fax +81-463-82-9627 |
| 2) | The tests were performed by the Mitsubishi Chemical Safety Institute Ltd., 14 Sunayama, Hasaki-machi, Kashima-gun, Ibaraki, 314-0255, Japan. Tel +81-479-46-2871 Fax +81-479-46-2874 |