2-methyl-p-phenylenediamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well-documented publication, comparable to guideline with deviation

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

(Mice were used instead of rats (preferred species) and the high dose group did not fulfill the recommended number of at least 20 dams/group)

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-p-phenylenediamine sulfate

EC Number:

210-431-8

EC Name:

2-methyl-p-phenylenediamine sulfate

Cas Number:

615-50-9

Molecular formula:

C7H10N2.H2O4S

IUPAC Name:

2-methyl-p-phenylenediamine sulphate

Constituent 2

Reference substance name:

2-methyl-p-phenylenediamine sulphate

EC Number:

228-871-4

EC Name:

2-methyl-p-phenylenediamine sulphate

Cas Number:

6369-59-1

IUPAC Name:

2-methylbenzene-1,4-diamine sulfate

Constituent 3

Reference substance name:

2, 5-toluenediamine sulfate

IUPAC Name:

2, 5-toluenediamine sulfate

Details on test material:

- Name of test material: 2,5-toluenediamine sulfate(2,5 TDS) - Substance type: Pure active substance- Physical state: Not reported- Stability under test conditions: Not reported- Storage condition of test material: Not reported

Test animals

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Breeding Laboratories (Wilmington, MA)- Age at study initiation: 60-90 d- Weight at study initiation: Not reported- Housing: Prior to mating, the females were housed in polypropylene in a group of 10/cage and males were housed individually in another room in polypropylene mouse cages. The mated female mice with vaginal plugs were divided into experimental and control groups and were caged in a group of

Administration / exposure

Route of administration:

subcutaneous

Vehicle:

other: Sterile, distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in sterile, distilled water. VEHICLE:- Dose volume: 1% bw (10 mL/kg) The volume was based on the weight of each mouse on the day the test substance was administered.- Concentration of test material in vehicle: 1.6, 3.2, 4.8 and 6.4 mg/mL for 16, 32, 48 and 64 mg/kg bw respectively.- Lot/batch no: Gibco, Grand Island, NY; Lot # Not reported

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: Cohoused - M/F ratio per cage: 1:2- Length of cohabitation: Males and females were housed together, and the following morning the mice with vaginal plugs were caged (

Duration of treatment / exposure:

Animals were treated on Day 6-15 of gestation

Frequency of treatment:

Animals were treated once daily

Duration of test:

Animals were sacrificed on Day 18 of gestation

No. of animals per sex per dose:

The numbers of dams treated were as follows: 31 (control), 27 (16 mg/kg/day), 26 (32 mg/kg/day), 31 (48 mg/kg/day) and 11 (64 mg/kg/day).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels used in the study were chosen on the basis of toxicity information and were far in excess of any exposure levels likely for the average person, even an individual handling this substance.- Rationale for animal assignment: The dams were divided into control and experimental groups such that body weight differences between groups were minimized.- Rationale for route of administration: Due to the deficiencies inherent in dermal applications, it was decided that the subcutaneous route would closely mimic the absorption mechanism for hair dyes in humans, while insuring consistent, quantitative uptake of the test substance.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: Not reported- Cage side observations included: MortalityDETAILED CLINICAL OBSERVATIONS: NoBODY WEIGHT: Yes - Time schedule for examinations: Body weight gain was recorded for the time periods of Days 6-17POST-MORTEM EXAMINATIONS: Yes - Sacrifice on: On Day 18 of gestation (by cervical dislocation)- Organs examined: Reproductive status was determined with emphasis on uterus, uterine horns and general condition of each conceptus.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: YesExaminations included:- Gravid uterus weight: No- Number of corpora lutea: No- Number of implantations: Yes - Number of resorptions: Yes; The number of resorptions was recorded. However, the study did not specify if they were early or late resorptions.- Other: The general condition of each conceptus was recorded. The staining of implantation sites in the uteri of apparently non-pregnant females was achieved through use of ammonium sulfide.

Fetal examinations:

- External examinations: Yes, live fetuses were weighed individually and checked for external malformations. - Fetal sex ratio: Yes, the sex of each fetus was determined by internal (surgical incision below navel) examination.- Dead/Alive fetus: Yes - Number of stunted fetus: Yes, live fetuses weighting

Statistics:

The litter was considered as the experimental unit for analysis of data regarding embryotoxicity and teratogenicity. Statistical significance of differences between groups was determined by the Mann-Whitney U test, while Jonckheere’s test was employed to test the significance of dose response relationships. Two-tailed tests were performed and p<0.05 was selected as the level of statistical significance.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yesDetails on maternal toxic effects:64 mg/kg/day was lethal to 9 of 11 pregnant mice. The 48 mg/kg/day dose was also toxic, as indicated by the deaths of 4 of 31 treated mice. Although none of the dose levels tested caused a significant reduction in weight gain during pregnancy, there was a significant trend (p<0.05) in that direction as the dose increased.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

32 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yesDetails on embryotoxic / teratogenic effects:Average fetal weight tended to decrease as the dose increased. There was a significant (p<0.05) decline in fetal weights at doses of 32, 48 and 64 mg/kg/day and therefore it was concluded that there was evidence of embryotoxicity. In contrast, there was no evidence of an effect on the percent of malformed fetuses, number of resorptions, number of fetal deaths and number of stunted fetuses at any dose group and thus the test substance was determined not to be teratogenic.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

>= 64 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

NOAEL

Effect level:

16 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Reproductive performance of 2,5-toluenediamine sulfate (2,5 TDS) in mice (Study # OP46771)

	Dose Group (mg/kg/day)
	Control	16	32	48	64
Number of dams receiving test material	31	27	26	31	11
Number of dams alive on Day 18	31	27	26	27	2
Number of dams pregnant*	24	25	20	23	2
Average weight gain (gm) during pregnancy (Days 6-17)	18.0	17.6	16.7	16.0	17.0
Total number of implants	295	324	253	270	30
Average number of implants'	12.3	13	12.7	11.7	15
Number of resorptions	28	38	19	20	3
Percent resorptions of total number of implants	9.5	11.7	7.5	7.4	10
Number of fetal deaths	7	4	9	4	0
Percent fetal deaths of total number of implants	2.4	1.2	3.6	1.6	0
Male/female-live fetuses	131/129	135/147	111/114	107/139	17/10
Number of stunted fetuses	2	0	0	6	0
Average number of live fetuses per dam	10.8	11.3	11.3	10.7	13.5
Average fetal weight** (g)	1.04±0.027	0.974±0.017	0.936±0.016	0.934±0.032	0.861±0.014

* = Included dams with resorptions

** = Dead and stunted fetus were excluded

 

Applicant's summary and conclusion

Conclusions:

2 -methyl-p-phenylenediamine sulphate (2, 5-toluenediamine sulfate) when administered subcutaneously to mated female CD-1 mice at 0, 16, 32, 48and 64 mg/kg/day was considered non-teratogenic and non-emryo toxic at 64 and 16 mg/kg/day respectively. The NOAEL for maternal toxicity, teratogenicity and embryotoxicity were 32 , 64 and 16 mg/kg/day respectively.

Executive summary:

The teratogenicity study of 2 -methyl-p-phenylenediamine sulphate (2,5-toluenediamine sulfate) was determined following OECD guideline 414 (Prenatal Developmental Toxicity Study).

This study was designed to evaluate the teratogenic effects of 2 -methyl-p-phenylenediamine sulphate when administered subcutaneously to mated female CD-1 mice once daily from Day 6-15 of gestation at dose levels of 16, 32, 48 and 64 mg/kg/day. Control group (0 mg/kg bw) were administered with sterile, distilled water (vehicle) only.

 

Male and nulliparous female mice ( 60-90 days old) were obtained from Charles River Breeding Laboratories (Wilmington, MA). and were cohoused by placing two females into each male's cage for breeding . Vaginal plugs (Day 1 of gestation) were observed for the proof of pregnancy. By Day 6 of gestation, the dams were divided into experimental and control groups such that body weight differences between groups were minimized and were caged in a group of </=10 dams/cage.

The test solution was prepared in sterile, distilled water. The test solution and the control were administered once a day in a volume equivalent to 1% bw (10 mL/kg) on Days 6 -15 of gestation. The volume was based on the weight of each mouse on the day the test substance was administered at the following dose levels:

0 (31 dams), 16 (27 dams), 32 (26 dams), 48 (31 dams) and 64 (11 dams) mg/kg/day.

All animals were observed for mortality and body weight gain of the dams was recorded for the gestation periods of Days 6-17. On Day 18 of gestation the mice were killed by cervical dislocation and their reproductive status was determined. The number of resorptions was recorded. Implantation sites in each uterine horn were counted and the general condition of each conceptus was recorded. The staining of implantation sites in the uteri of apparently nonpregnant females was performed with ammonium sulfide.

Live fetuses were weighed individually, sexed and checked for external malformations. Number of stunted fetus (live fetuses weighing </=0.5 g or less than two thirds the mean of their larger littermates) were determined. At least one-third of the fetuses of each litter, as well as all stunted fetuses and those having external malformations, were examined for visceral alterations. The bodies of all fetuses were then processed for skeletal examination. The heads of those fetuses subjected to visceral examination (with the exception of any fetuses which had external head malformations) were cut off at the base and prepared for free-hand sectioning.

The litter was considered to be the experimental unit for analysis of data regarding embryotoxicity and teratogenicity. Statistical significance of differences between groups was determined by the Mann-Whitney U test, while Jonckheere's test was employed to test the significance of dose response relationships. Two-tailed tests were performed and p<0.05 was selected as the level of statistical significance.

In this study, treatment at 64 mg/kg/day resulted in mortality of 9 of 11 pregnant mice. The 48 mg/kg/day dose was also toxic, as indicated by the deaths of 4 of 31 treated mice. Although none of the dose levels tested caused a significant reduction in weight gain during pregnancy, there was a significant trend (p<0.05) in that direction as the dose was increased.

Average fetal weight tended to decrease as the dose was increased. A significant (p<0.05) decline in fetal weights at dosages of 32, 48 and 64 mg/kg/day was observed and therefore it was concluded that there was evidence of embryotoxicity. No evidence of an effect on the average percentage of malformed fetuses was observed at any of the dose levels and thus the test substance was determined not to be teratogenic.

Based on the results above, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity, teratogenicity and embryotoxicity were 32 , 64 and 16 mg/kg/day, respectively.

This teratogenicity study is classified as acceptable, and satisfies the guideline requirements of the OECD 414 method.