(p-ammoniophenyl)bis(2-hydroxyethyl)ammonium sulphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The multigeneration reproduction study was designed to evaluate the reproductive toxicity of two formulation hair dyes containing N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate in Charles River CD rats.  Dermal application of P21 formulation containing 1% N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate mixed  with equal volume of hydrogen peroxide and dermal application of P22 formulation containing 0.5% of test substance to Charles River CD rats over three generations did not induce any treatment related effects on reproductive parameters in any of the generations. Additionally, a OECD 408 study was performed in which rats were treated orally for 90 fays to the test substance. Reproductive apparatus analysis was perfomed. No effect was observed on sperm motility and vaginal cytology. The NOAEL was defined as 20 mg/kg bw/day.

Based on the results of the available studies, the registered substance N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate was Not Classified for Toxicity to Reproduction.

Link to relevant study records

Reference

Reference 1

Endpoint:

multi-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From Oct. 17, 1974 to Sept. 24, 1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed scientific principles/standards

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The multigeneration reproduction study was designed to determine the effect of test substance on the reproductive parameters of three generations (F1 generation: F1a, F1b; F2 generation: F2a, F2b; F3 generation: F3a, F3b and F3c). The test substance was administered dermally at a dosage level of 0.5 mL/rat, twice a week continuous through growth, mating, gestation and lactation to the weaning of F1b, F2b and F3c litters of respective generations. The parental rats and pups were observed for general behavior, body weight, survival, fertility index, gestation index and viability index throughout the study. F1b parental rats and F3b weanling rats were subjected to gross and histopathology.

GLP compliance:

no

Remarks:

(pre-GLP)

Limit test:

no

Species:

rat

Strain:

other: Charles River CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington
- Age at study initiation: P: 100 d; F1: 100 d; F2: 100 d
- Weight at study initiation: (P) Males: 97-218 g; Females: 68-172 g
- Fasting period before study: Not reported
- Housing: Rats were housed individually in hanging wire mesh cages
- Use of restrainers for preventing ingestion (if dermal): Not reported
- Diet: Ground Purina Chow; ad libitum
- Water: Ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
No information on environmental conditions is provided in the study. However, it is reported that the animals were maintained in a temperature, humidity and light controlled room.

EXPERIMENT INITIATION DATE: Oct. 17, 1974
EXPERIMENT COMPLETION DATE: Sept. 24, 1976

Route of administration:

dermal

Vehicle:

water

Details on exposure:

TEST SITE: The hair on the neck and back of each rat was clipped with an electric clipper (at least 24 h prior to test substance application) as necessary during the study. The control rats were clipped in the same regimen, but no test substance was applied.
APPLICATION: The formulations were applied as evenly as possible to assure skin contact and to avoid run off. Skin irritation was minimized by applying the dyes over adjacent areas of skin on successive application days. Prior to treatment, the formulation 1 (P 21) was thoroughly shaken and mixed with equal volume of 6% hydrogen peroxide. This mixture was used within 15 min of preparation. Formulation P22 was applied directly from the bottle.

REMOVAL OF TEST SUBSTANCE: The details on removal of test substance are not provided in the study report.

TEST MATERIAL
- Amount(s) applied: 0.5 mL/rat/twice a week
- Concentration: (i) P21 formulation containing 1% N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate mixed with equal volume of 6% hydrogen peroxide (ii) P22 formulation containing 0.5 % N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate
- Constant volume or concentration used: No, initial dose volume was 0.2 mL/animal was increased by increments of 0.1 mL/application weekly until reaching 0.5 mL/application.

USE OF RESTRAINERS FOR PREVENTING INGESTION: No

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 15 days
- Proof of pregnancy: Presence of sperm in vagina or the copulatory plug was referred as Day 0 of pregnancy.
- After successful mating each pregnant female was caged (how): The females were placed in individual nesting boxes (with ground cob bedding) and permitted to deliver their young naturally.
During mating each dye was allowed to dry on the rats' skin before the rats were returned to their cages with mates.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

The test substance was administered continuously through growth, mating, gestation and lactation to the weaning of F1b, F2b and F3c litters of respective generations.

Frequency of treatment:

Twice weekly

Details on study schedule:

F1 generation:
- Application of the dyes was made with rats in individual cages until they reached 100 d of age. After this, the rats were paired on a one-to-one basis, care being taken to ensure that littermates were not paired for mating.
- Mating continued for 15 d and the females were smeared daily to determine the presence of sperm in the vagina. The females were permitted to deliver.
- After weaning of the F1a litters, the F0 parents were reduced in number to 20 males and 20 females for each of the 7 groups.
- Following a 10 d rest, the remaining F0 parents were rebred to produce the F1b litters. Mating was as described for the production of F1a generation.

F2 generation:
- 20 males and 20 females were selected from F1b litters in each group and these became the F1 parents for next generation.
- Pups were removed singly from the individual litters. F1 parental rats were individually housed and treated as described above for the F0 generation.
- At approx. 100 days of age, the rats were mated, avoiding brother-sister pairings for the production of F2a and F2b litters.

F3 generation:
- 20 males and 20 females were selected from the F2b litters to be parents for next generation. F2b parents were mated for the production of F3a, F3b and F3c litters.

Dose / conc.:

1 other: 1% N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate of mixed with equal volume of 6% hydrogen peroxide (P21 formulation)

Remarks:

nominal concentration

Dose / conc.:

0.5 other: % N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate (P22 formulation)

Remarks:

nominal concentration

No. of animals per sex per dose:

F0 generation: 40 animals/sex/dose
F1 and F2 generation: 20 animals/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Not reported
- Rationale for animal assignment: The rats were selected such that the initial group mean body weight for each sex was similar. Littermates were evenly distributed among the groups.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations included: General behavior and appearance

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Weekly

- OTHER: Reproductive parameters were evaluated to determine fertility index, gestation anomalies, effects on parturition and lactation.

Sperm parameters (parental animals):

Parameters examined in [F1b, F2b and F3b] male parental generations: Testes, seminal vesicles were examined at necropsy.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes, on Day 4 of lactation
- If yes, maximum of [10]pups/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
- Number of live pups on Day 0, 4 and 14
- Mean weight of pups on Day 0, 4 and 14
- Pups were counted, sexed and weighed individually on Day 21 of lactation
- Pups were observed for signs of pharmacotoxic effects

GROSS EXAMINATION OF DEAD PUPS: Not reported

Postmortem examinations (parental animals):

SACRIFICE
- The F0 parents were sacrificed and discarded following the weaning of the F1b litters.
- After weaning of the F2b litters, 5 male and 5 female F1b parental rats from each group were sacrificed, gross necropsy performed and tissues collected for microscopy.
- After weaning of the F3a and F3c litters, the young were sacrificed and discarded.
- Rats which died during the course of study were subjected to gross necropsy examination.

GROSS NECROPSY
- At necropsy, the following tissues and organs of F1b parental rats were examined:
Adrenal, lungs, skin, aorta, lymph node (mesenteric), spleen, colon, stomach, esophagus, ovaries, testes, eyes, pancreas, thymus, heart, peripheral nerve, thyroid, ileum, prostate, trachea, jejunum, salivary gland, urinary bladder, kidney, seminal vesicles, uterus, liver and skeletal muscle.
HISTOPATHOLOGY
- The following tissues from 5 male and 5 female F1b parental rats were prepared and examined for microscopic evaluation:
Adrenals, liver, thyroid, colon, lungs, urinary bladder, heart, ovaries, uterus, ileum, spleen, skin, jejunum, stomach, kidney and testes.
- Stain used: Hematoxylin and eosin stain

Postmortem examinations (offspring):

SACRIFICE
- Representative pups of F3b litters (one weanling from each litter) were sacrificed with chloroform at weaning, necropsied and subjected to histopathological evaluation.
- Numbers of weanling examined varied from 3 to 17 per group.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- At necropsy, the following tissues and organs of selected F3b litters were examined:
Adrenal, lungs, skin, aorta, lymph node (mesenteric), spleen, colon, stomach, esophagus, ovaries, testes, eyes, pancreas, thymus, heart, peripheral nerve, thyroid, ileum, prostate, trachea, jejunum, salivary gland, urinary bladder, kidney, seminal vesicles, uterus, liver and skeletal muscle.
HISTOPATHOLOGY
The following tissues from selected F3b litters were prepared and examined for microscopic evaluation:
Adrenals, liver, thyroid, colon, lungs, urinary bladder, heart, ovaries, uterus, ileum, spleen, skin, jejunum, stomach, kidney and testes.
- Stain used: Hematoxylin and eosin stain

Statistics:

- All statistical analyses compared the treatment groups with the control group.
- Fertility indices and gestation at 4, 14 and 21-day survival indices were compared using the Chi-square test criterion with Yates correction for 2 x 2 contingency tables and/or Fisher's exact probability test as described by Siegel to judge significance of differences.
- Gestation 4, 14 and 21-day survival indices were also compared by the Mann-Whitney U-test as described by Siegel and Weil to judge significance of differences.
- Mean numbers of live born pups per litter and mean litter weights at 0, 4 and 14 days were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances).

Reproductive indices:

Fertility index was determined.

Offspring viability indices:

The viability index of offspring was determined.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY:

F0 generation:
- The test groups elicited skin reactions consisting of mild scabbing, fissuring, atonia and leathery texture intermittently throughout the treatment period, whereas, the control group elicited virtually no skin effects.
- All rats of the F0 generation in control and test groups evinced, on occasion soft stools, diarrhea some respiratory congestion and nasal discharge. - No pharmacotoxic signs were observed that could be attributed to the dermal application through the first (F0) generation.

BODY WEIGHT AND FOOD CONSUMPTION:
- The mean body weights, weight ranges for the parental rats administered with P21 and P22 were comparable with the control group, for both sexes, in each generation.
- Similarly, there were no significant differences between food consumption data for test and control groups.

REPRODUCTIVE PERFORMANCE:
- The performance of the F0, F1 and F2 parental rats showed no differences between the control and test groups as far as fertility and live birth indices were concerned.

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation:
- The F1, second generation, parental rats of the test groups elicited signs of a low grade dermal irritation.
- Other alterations in appearance and behavior were seen with equal frequency between test and control group and they included occasional signs of respiratory congestion, alopecia, soft stools, occasional diarrhea and intermittent signs of hypoactivity.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- The mean body weights, weight ranges for the parental rats administered with P21 and P22 were comparable with the control group, for both sexes, in each generation.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- No gross pathologic lesions which were considered related to test substance application were seen in any F1b parental rats which were sacrificed and necropsied. No treatment related lesions were seen in any of the rats which died during the course of study.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- No treatment related microscopic lesions were seen in any tissues examined from F1b parental rats.

Reproductive function: oestrous cycle:

no effects observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathologic lesions which were considered related to test substance application were seen in any F1b parental rats which were sacrificed and necropsied. No treatment related lesions were seen in any of the rats which died during the course of study.

Histopathological findings:

no effects observed

Description (incidence and severity):

No treatment related microscopic lesions were seen in any tissues examined from F1b parental rats.

- The litter size and body weights of the young were similar for the control and the test groups.
- Survival and body weights for offspring of treated females were not different from control values for the same parameters during the lactation period, until weaning.
- There were certain statistically significant differences at some instances, however these were not indicative of biological differences.
- No treatment related gross or microscopic pathologic lesions were seen in any F3b weanling rats which were sacrificed and necropsied.

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- As with the previous generations, a variety of incidental findings that were not related to test substance application were found in each group of F2 generation parental rats.
- During Week 61, sialadenitis was observed for some rats in each of the test groups and in the control groups. The sialadenitis regressed by Week 63 but it was followed in subsequent weeks by the appearance of an increased incidence of respiratory congestion in rats of both control and test group. During the two weeks prior to the establishment of sialadenitis essentially no evidence of respiratory congestion was noted. Starting immediately after regression of the sialadenitis there was a notable increase in respiratory congestion. The latter was considered to be a consequence of the debilitating effect of sialadenitis on the rats, which increased their susceptibility to disease.
- The respiratory congestion persisted in the F2 parents during the production of successive litters, but this condition was not considered a significant factor in the reduction of fertility indices.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights, weight ranges for the parental rats administered with P21 and P22 were comparable with the control group, for both sexes, in each generation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Similarly, there were no significant differences between food consumption data for test and control groups.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The performance of the F0, F1 and F2 parental rats showed no differences between the control and test groups as far as fertility and live birth indices were concerned.
The F2 parents, however, had markedly reduced fertility indices for three separate matings to produce F3a , F3b and F3c litters. There were no significant differences between the control and test groups with regard to fertility.
- Therefore, it was not considered that the dyes P-2l and P-22 caused the reduction in fertility

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

SPECIAL STUDY:

- Of the 20 female rats that failed to produce offspring in all F3 litters, 45% of these females had microscopically evident reproductive tract changes which may have contributed to their unsuccessful reproductive performance.

- Of the 20 male rats that failed to produce young, 15% of these males had reproductive tract lesions which might have contributed to their unsuccessful reproductive performances.

- These lesions were observed in control as well and therefore were considered spontaneous and unrelated to test substance.

Conclusions:

Dermal application of P21 formulation containing 1% N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate mixed with equal volume of hydrogen peroxide and dermal application of P22 formulation containing 0.5% of test substance to Charles River CD rats over three generations did not induce any treatment related effects on reproductive parameters in any of the generations.

Executive summary:

The  multigeneration reproduction study was designed to evaluate the reproductive toxicity of two formulation hair dyes containing N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate in Charles River CD rats. 

The hair dyes were applied dermally at a dosage level of 0.5 mL/rat to Charles River CD rats through three generations at following concentrations:

P21 formulation containing 1% N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate mixed with equal volume of 6% hydrogen peroxide

P22 formulation containing 0.5 % N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate

The experimental animals (parental generation) were sourced from Charles River Breeding Laboratories, Wilmington at age of 100 d and were housed individually in hanging wire mesh cages. The animals were maintained in a temperature, humidity and light controlled room andwere allowed free access to food and water throughout the study.

The hair on the neck and back of each rat was clipped with an electric clipper (at least 24 h prior to test substance application) as necessary during the study. The control rats were clipped in the same regimen, but no test substance was applied. Prior to treatment, the Formulation P 21 was mixed with equal volume of 6% hydrogen peroxide. This mixture was used within 15 min of preparation. Formulation P22 was applied directly from the bottle. The formulations were applied as evenly as possible to assure skin contact and to avoid run off.

The dyes were administered dermally at a dosage level of 0.5 mL /rat, twice a week. The initial dosage level was 0.2 mL per application, which was increased by increments of 0.1 mL per application weekly until reaching 0.5 mL per application. Application of the dyes was made with rats in individual cages until they reached 100 days of age. After this, animals were subsequently mated on a one to one basis. The pregnant females were placed in individual nesting boxes and were permitted to deliver their young naturally. After weaning of F1a litters, the F0 parents were reduced in number to 20 males and 20 females. Following a 10 day rest, the remaining F0 parents were rebred to produce F1b litters.

Similarly, F1b litters were selected to be parents of the next generation. The animals were mated at approx. 100 d of age for the production of F2a and F2b litters. Following the procedures above, F2 parents were selected from F2b litters for the production of F3a, F3b and F3c litters. Treatment was continuous through growth, mating, gestation and lactation to the weaning of F1b, F2b and F3c litters of respective generations.

The animals were observed for mortality, general behavior and appearance daily. Detailed clinical observations were performed weekly. Individual body weights and sex group food consumption were recorded weekly. Reproductive parameters were evaluated to determine fertility index, gestation anomalies, effects on parturition and lactation. The pups were counted and weighed as a litter on Day 0, 4 and 14 of lactation. The pups were counted, sexed and weighed individually on Day 21 of lactation. Indices for live birth and survival, to weaning, were calculated. The pups were observed for signs of pharmacotoxic effect. F1b parental rats and F3b weanlings were subjected to gross and histopathological evaluation.

No changes considered to be related to the dyes were seen for the parental rats or pups in general behavior and appearance, body weights or survival. However, the treated rats did show some mild skin reactions intermittently throughout the study. The fertility, gestation and viability indices were comparable for control and treated groups. No treatment related gross or microscopic pathologic lesions were seen in any F1b parental rats or F3b weanling rats which were sacrificed and necropsied. No treatment related gross pathologic lesions were seen at necropsy in any rats which died during the course of study.

Based on above, dermal application of P21 formulation containing 1% N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate mixed with equal volume of hydrogen peroxide and P22 formulation containing 0.5% of test substance to Charles River CD rats over three generations did not induce any treatment related effects on reproductive parameters in any of the generations.

Reference 2

Endpoint:

fertility, other

Remarks:

90 days repeated toxicity study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12 March 2004 To 30 November 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 408 Repeated Dose 90-Day Oral Toxicity Study in Rodents

Version / remarks:

In this Repeated dose -90 day oral toxicity study, some reproductive parameters as fertility were evaluated

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot No. A203G157
- Expiration date of the lot/batch: not specified
- Purity test date: 20 November 2003

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: homogeneity : Mean results of the homogeneity analyses from the top, middle, and bottom locations of the 0.1- and 2.0-mg/mL concentrations ranged from 96.6 to 97.0% and 98.5 to 99.5% of theoretical, respectively, indicating that the mixing procedure resulted in homogeneous preparations.

Stability : Dose confirmation mean results for all concentrations ranged from 96.6 to 98.5%, 96.8 to 99.5%, 97.7 to 99.7%, 98.6 to 99.5%, and 74.7 to 90.8% of theoretical for Weeks 1 (initial), 2, 5, 9, and 13, respectively. Results of the Week 13 analysis of the 0.1-mg/mL concentration did not meet acceptance criteria (within 15% of theoretical).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
For each dose formulation, a volume of vehicle was added to the required amount of GTS03849 and mixed on a magnetic stir plate and stir bar until the formulation appeared to be a uniform suspension. Additional vehicle was added to achieve the final volume. The formulations were sampled (if required), then transferred to individual amber glass containers for daily use and stored in a refrigerator set to maintain 2 to 8°C, protected from light.

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: the animals were approximately 6 to 7 weeks old,
- Weight at study initiation:their body weights were ranged from 204 to 299 g for the males and 153 to 194 g for the females
- Fasting period before study: not specified
- Housing: The animals were housed individually in suspended, stainless-steel cages.
- Diet (e.g. ad libitum):Certified Rodent Diet (#8728C, Harlan Teklad) was available ad libitum
- Water (e.g. ad libitum):Water was available ad libitum. Water samples are routinely analyzed for specified microorganisms and environmental contaminants
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%):30 to 70% Relative humidity
- Air changes (per hr): 10 or greater air changes/hour
- Photoperiod (hrs dark / hrs light):12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 15 March 2004 To: 19 July 2004

Route of administration:

oral: gavage

Vehicle:

other: 0.2% erythorbic acid in reverse osmosis water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Vehicle formulations were prepared weekly for this study. The vehicle components were reported to be 100% pure for the purpose of dosage calculations. For the preparation of the
vehicle, a portion of reverse osmosis (RO) water was transferred to a labeled container. The required amount of erythorbic acid was transferred to the RO water while mixing using a
magnetic stir plate and stir bar until the erythorbic acid was dissolved. The preparation was then brought to final volume with RO water.
Test article formulations were prepared approximately weekly for this study. The test article was 100% pure for the purpose of dosage calculations. For each dose formulation, a volume of vehicle was added to the required amount of registered substance and mixed on a magnetic stir plate and stir bar until the formulation appeared to be a uniform suspension. Additional vehicle was added to achieve the final volume. The formulations were sampled (if required), then transferred to individual amber glass containers for daily use and stored in a refrigerator set to maintain 2 to 8°C, protected from light.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: 0, 0.1, 0.4, 2.0 mg/mL
- Amount of vehicle (if gavage): 10mL/kg
- Lot/batch no. (if required): 11719CA Sigma-Aldrich Co.
- Purity: 99.9%

Details on mating procedure:

not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Mean results of the homogeneity analyses from the top, middle, and bottom locations of the 0.1- and 2.0-mg/mL concentrations ranged from 96.6 to 97.0% and 98.5 to 99.5% of theoretical, respectively, indicating that the mixing procedure resulted in homogeneous preparations. Stability analyses generated in Covance 6114-461 indicated that the formulations were stable for the concentration range used and under conditions of use for the study.

Dose confirmation mean results for all concentrations ranged from 96.6 to 98.5%, 96.8 to 99.5%, 97.7 to 99.7%, 98.6 to 99.5%, and 74.7 to 90.8% of theoretical for Weeks 1 (initial), 2, 5, 9, and 13, respectively. Results of the Week 13 analysis of the 0.1-mg/mL concentration did not meet acceptance criteria (within 15% of theoretical).

Duration of treatment / exposure:

91 days

Frequency of treatment:

Once daily

Details on study schedule:

no mating was performed

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

4 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 animals per sex were sued for control and high dose group, 15 animals per sex were used for low and mid dose level group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses selected for this study were based on the results of a 14-day range-finding study (Covance 6114-469). The goal of the dose level selection was to establish satisfactory safety margins for various levels of potential human exposure to the test article. Ideally, none of the dose levels should produce significant adverse effects. The oral route was chosen to maximize the potential to identify possible health hazards that may be associated with repeated exposure.
- Rationale for animal assignment (if not random): Animals were assigned to treatment groups using a computerized blocking procedure designed to achieve body weight balance with respect to treatment groups.

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS: Yes, each animal was observed for mortality, abnormalities and signs of pain or distress; findings were recorded as they were observed.
- Time schedule: Twice daily (a.m. and p.m.)

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations included: Abnormal findings were recorded

DETAILED CLINICAL OBSERVATIONS: Yes, these observations were made outside the home cage and included, but were not limited to, changes in skin, fur, eyes, and mucous membranes; occurrences of secretions and excretions; and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in posture and reactivity to handling and the presence of clonic or tonic movements, stereotypes (e.g., excessive grooming, circling), or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded. Changes in gait were assessed by allowing the animal to walk freely to allow evaluation of gait. Additional findings were recorded as they were observed.
- Time schedule: Prior to treatment, on Day 1, weekly thereafter, and on the day of each scheduled sacrifice

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded twice prior to treatment, on the first day of treatment, and weekly thereafter.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes, Animals were examined by a board-certified veterinary ophthalmologist using an indirect ophthalmoscope and a slit lamp. The eyes were dilated with a mydriatic agent prior to examination.
- Time schedule for examinations: Prior to treatment in a sequential manner and during Wk 13 in random order. The ophthalmic examinations were done on a day other than that scheduled for the expanded clinical observations.
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes, blood samples for were collected from a jugular vein of animals.
- Time schedule for collection of blood: At each scheduled sacrifice (on Day 92 and on Day 120)
- Anaesthetic used for blood collection: No
- Anticoagulants used: Yes potassium EDTA (for hematology) and sodium citrate (for coagulation parameters)
- Animals fasted: Yes (overnight)
- How many animals: All surviving animals
- Parameters examined: Red blood cell (erythrocyte) count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, white blood cell (leukocyte) count, differential blood cell count, blood smear, reticulocyte count, prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes, blood samples for were collected from a jugular vein of unanesthetized animals.
- Time schedule for collection of blood: At each scheduled sacrifice (on Day 92 and on Day 120)
- Animals fasted: Yes (overnight)
- How many animals: All surviving animals
- Parameters examined: Glucose, urea nitrogen, creatinine, total protein, albumin, globulin, albumin/globulin ratio, cholesterol, total bilirubin, alanine aminotransferase, alkaline phosphatase, gamma glutamyl transferase, aspartate amino transferase, calcium, inorganic phosphorus, sodium, potassium and chloride.

URINALYSIS: Yes, urine specimens were collected on wet ice
- Time schedule for collection of urine: At each scheduled sacrifice (on Day 92 and on Day 120).
- Metabolism cages used for collection of urine: Yes, individual urine collection cages were used
- Animals fasted: Yes (overnight)
- Parameters examined: Specific gravity, pH, protein, glucose, ketones, bilirubin blood, urobilinogen, volume, microscopic examination of sediment, appearance/color and urine chemistry (Sodium, potassium, chloride, sodium excretion, potassium excretion and chloride excretion).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Hand-held and open-field expanded clinical observations were done once prestudy and during Wk 4, 8, and 13 (in random order); elicited behavior observations were done once prestudy and during Wk 13 (in random order); and motor activity data were collected prestudy and during Wk 13. Expanded clinical observations and motor activity testing were done on a day other than that scheduled for the weekly detailed clinical observations
- Dose groups that were examined: 10 animals/sex/group (same animals were tested for all the three battery of functions)
- Battery of functions tested: Hand-held and open-field expanded clinical observations, elicited behavior observations, and motor activity. Additional findings were recorded as they were observed.

Oestrous cyclicity (parental animals):

Vaginal Cytology
All surviving females were evaluated daily for 21 consecutive days beginning after Week 10 (Days 71 through 91). Daily vaginal smears were prepared and examined to evaluate the stage of the estrous cycle for each female. For each animal, the estrous stage was documented each day for the 21 consecutive days. Examinations were done at approximately the same time each day and at a time corresponding to one that was after the completion of the expanded clinical observations.

Sperm parameters (parental animals):

Male Reproductive Assessment

Sample Collection
For motility and morphology assessment, the right vas deferens was excised and immediately placed in a petri dish containing 10 mL of a 1% bovine serum albumin dissolved in phosphate-buffered saline. The solution was prewarmed to approximately 38°C. A 3 to 4-minute period (minimum to maximum period, respectively) was allowed for the sperm to swim out.
The right epididymis was removed and divided in half by cross-sectioning through the middle. The tail end caudal section was placed on dry ice, transferred to PAI, and stored frozen at approximately -60 to -80°C for total sperm count. The left epididymis and remaining portion of the right epididymis were collected as described under Tissue Preservation.

Sperm Motility Evaluation
Following the swim-out period, a sperm sample was collected using a 100-micron-deep cannula and immediately loaded into a prewarmed stage of the Hamilton Thorne IVOS automated sperm analyzer. Five fields/animal were selected and stored as digital images. These images were analyzed for percent motility and transferred to optical media for permanent storage.

Sperm Morphology Evaluation
Sperm morphology was assessed with two slides of sperm stained with eosin for each male. A minimum of 200 sperm cells were evaluated.

Total Sperm Count Determination
At Pathology Associates, each frozen epididymis was thawed, and the caudal section was trimmed, weighed, and homogenized. A 100-microliter sample of the suspension was stained
with a dye that uniquely stains the sperm heads. A sample of the stained sperm was placed into a20-micron-deep glass slide and loaded into the analyzer. Twenty fields/animal were counted and reported, and adjusted for caudal epididymidal weight (taken following thawing at PAI).

Postmortem examinations (parental animals):

SACRIFICE: On Day 92, up to 15 animals/sex/group were fasted overnight, anesthetized with carbon dioxide, weighed, and exsanguinated. On Day 120, (after 4 Wk recovery), all surviving animals in Groups 1 and 4 were fasted overnight, anesthetized with carbon dioxide, weighed, and exsanguinated.

GROSS PATHOLOGY: Yes, each animal sacrificed, or died during the study was necropsied. The necropsy included an examination of the external features of the carcass; external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues.
- Tissues from each animal (when present) were preserved in 10% neutral-buffered (except testis-preserved in Bouin’s fixative): Adrenal (2), aorta, brain (cerebrum, cerebellum, and medulla), cecum, cervix, colon (proximal and distal (2)), duodenum, epididymis (2), esophagus, eye (2), femur with bone marrow (articular surface, of the distal end), Harderian gland, heart, ileum (including Peyer’s patch), jejunum, kidney (2), lacrimal gland (exorbital), liver, lung with mainstem bronchi, lymph node (mandibular and mesenteric), mammary gland (females), nasal turbinates, ovary (2), pancreas, pituitary gland, prostate, rectum, salivary gland (mandibular (2)), sciatic nerve, seminal vesicle (2), skeletal muscle (thigh), Skin, spinal cord (cervical, thoracic, and lumbar), spleen, sternum with bone marrow, stomach (nonglandular and glandular), testis (2), thymus, thyroid with parathyroid, tissues with macroscopic changes, or alterations (i.e., gross lesions), tongue, trachea, urinary bladder, uterus with uterine horns vagina and zymbal’s gland.

HISTOPATHOLOGY: Yes, preserved tissues from each animal in the control and high-dose groups at the terminal sacrifice and those that died during the study were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Macroscopic lesions were also examined microscopically from each animal in the low- and mid-dose groups.
Testes and epididymides from each terminal sacrifice male in the control and high-dose groups were examined microscopically by a board-certified veterinary pathologist:
- Following collection from each male at each scheduled sacrifice, the epididymis was separated from the testes. After adequate fixation in 10% neutral buffered formalin, each testis was sectioned transversely in half, and the cranial pole was sectioned for routine embedding in paraffin, histologic processing, and staining with hematoxylin and eosin. The remaining testicular tissue was stored in 70% ethyl alcohol.
- The epididymides were sectioned longitudinally and processed routinely for histologic evaluation. This longitudinal section should ensure that the head and mid portion of the epididymides were evaluated histologically. The remaining portions of the epididymides were stored in 10% neutral-buffered formalin.

PATHOLOGY PEER REVIEW:
A peer review of microscopic findings was performed by a board-certified veterinary pathologist at Pathology Associates (PAI). The peer review was performed according to the reviewer’s standard operating procedures.

Statistics:

The following statistical methods were used to analyze the body weight, body weight change, food consumption, motor activity, grip strength, nociceptive reflex, continuous clinical pathology, and organ weight data.
• Levene’s test (Levene, 1960; Draper and Hunter, 1969) was done to test for variance homogeneity. In the case of heterogeneity of variance at p < 0.05, rank transformation was used to stabilize the variance. Comparison tests took variance heterogeneity into consideration.
• One-way analysis of variance [(ANOVA, (Winer, 1971a)] was used to analyze data.
• If the ANOVA was significant (p < 0.05), Dunnett’s t-test (Dunnett, 1955, 1964) was used for control versus treated group comparisons.
The length of an estrus cycle for individual animals was determined by counting the number of days from a recording of estrus (including this first day of estrus) up to (but not including) the next recording of estrus that occurs after metestrus, diestrus, or proestrus are recorded. If estrus was not observed in an apparent cycle, then a metestrus recording that was preceded by either a diesturs or proestrus was considered the beginning of the cycle or a diestrus recording that was preceded by a proestrus was considered the beginning of the cycle. A full cycle ended with the day prior to a recording of estrus, with the exception that a recording of proestrus on the last (21st) day of estrus evaluations was considered the end a full cycle. This last proestrus recording was included as the final day of the last estrus cycle. The mean estrus cycle duration

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean number of days in each stage and the mean estrus cycle duration were comparable
across groups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The mean percent sperm motility, caudal epididymal sperm count, and sperm morphology were
not affected by treatment with GTS03849 at dose levels of 1, 4, and 20 mg/kg/day. No
biologically meaningful differences were observed between the study groups.

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY: One Group 1 (vehicle control) male died on Day 72 from causes unrelated to treatment (severe, acute pyelonephritis). All other animals survived in apparent good health until scheduled sacrifice.
- No treatment-related clinical observations were noted during the treatment or recovery phase of the study. The few clinical observations noted for surviving animals (malocclusion, missing teeth, barbering, oral discharge, few feces, red haircoat, and sores or scabs) were unrelated to dose, were of small incidence and short duration, and therefore, were not considered treatment related.

BODY WEIGHT AND WEIGHT GAIN: Mean body weights and body weight changes of treated animals were comparable with controls throughout the treatment and recovery periods.

FOOD CONSUMPTION: No treatment-related effects on food consumption were noted during treatment and recovery.

OPHTHALMOSCOPIC EXAMINATION: There were no treatment-related ophthalmic observations

HAEMATOLOGY: Very few statistically significant or otherwise notable differences for clinical pathology test results were observed between the control and treated animals. None of the differences in the haematology parameters were attributable to treatment with test substance.

CLINICAL CHEMISTRY: Statistically significantly higher triglyceride concentration was observed in mid dose females at Wk 14. However, this variation was considered incidental, as the difference was extremely small (i.e., only 6 mg/dL), males were unaffected, and there were no correlative findings. The difference for triglyceride concentration was consistent with normal biological variation.

URINALYSIS: None of the differences in the urinalysis parameters were attributable to treatment with test substance.

NEUROBEHAVIOUR: No test substance-related effects on neurobehavioral assessment tests were apparent during treatment. Motor activity was significantly decreased at the 30-40 and 0-40 minute intervals for high dose group females. However, because values recorded during acclimation were also lower for high dose group females compared with controls and because a similar effect was not seen in males, these decreases were not considered to be toxicologically important.

ORGAN WEIGHTS: Terminal organ weight and organ to body weight ratio were not affected by treatment. Decreased mean absolute testicular and thymic weight values were observed in high dose group recovery males. However, there was no histological correlate to support this finding and were not attributed to the treatment.

GROSS PATHOLOGY: No treatment related differences were observed in macroscopic findings.

HISTOPATHOLOGY: NON-NEOPLASTIC: All histological findings were of the kind commonly found in laboratory rats of age and strain used in the study or were of singular occurrence or distributed (scattered) randomly among the groups without relation to dosage.

OTHER FINDINGS:
- Vaginal Cytology: The mean number of days in each stage and the mean estrus cycle duration was comparable across groups.
- Male Reproductive Assessment: The mean percent sperm motility, caudal epididymal sperm count, and sperm morphology were not affected by treatment. No biologically meaningful differences were observed between the study groups.

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Critical effects observed:

no

Remarks on result:

not measured/tested

Critical effects observed:

no

Remarks on result:

not measured/tested

Critical effects observed:

no

Reproductive effects observed:

no

Table1 :Sperm analysis parameters

	Group	1	2	3	4	Group (Recovery )	1	4
	Dose	0	1	4	20	Dose	0	20
Motility	Mean	83	78	87	82	Mean	85	80
	SD	18	19	10	11	SD	8	27
	N	14	14	15	15	N	5	5
Epididymal count (million sperm/gram)	Mean	904	833.2	965.8	918.6	Mean	464.4	390.6
	SD	110.6	323.7	205.1	162.1	SD	90.3	65.3
	N	14	15	15	15	N	5	5
SpermMorphology(%abnormal)	Mean	0.5	0.5	0.4	0.5	Mean	0.5	0.2
	SD	0.6	0.8	0.7	0.7	SD	0.7	0.3
	N	14	15	15	15	N	14	15

 

Conclusions:

Based on the results of the study, the No Observed Adverse Effect Level (NOAEL) following oral gavage administration of N,N-bis (2-hydroxyethyl)-PPD Sulf to rats at doses of 0, 1, 4, or 20 mg/kg/day for 91 d was determined to be 20 mg/kg/day. No toxicity on reproductive apparatus or fertily was observed.
 

Executive summary:

Subchronic toxicity of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate was performed following OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents). Analysis on fertility parameters as sperm motility on male and vaginal cytology. Hence, this study was considered to be relevant for toxicity to reproduction assessment.

The study was designed to evaluate the toxicity of the test substance, when administered daily at dose levels of 0, 1, 4 and 20 mg/kg bw/day via oral gavage to Crl:CD(SD)IGS BR rats for 91 d and to assess the reversibility of any effects after a 4 Wk recovery period. A total of 140 Crl:CD(SD)IGS BR rats (70/sex) of 6-7 Wk age (source: Charles River Laboratories, Portage, Michigan), weighing 204-299 g (males) and 153-194 g (females) were housed individually, in suspended stainless-steel cages. The animals were maintained under standard laboratory conditions (temperature: 19-25°C, relative humidity: 30-70%, 10 or greater air changes/h, 12 h light/12 h dark cycle per d).

The animals were observed twice daily (a.m. and p.m.) for mortality, abnormalities, and signs of pain or distress. Cage side observations were made for each animal once daily. Detailed clinical observations were performed prior to treatment, on Day 1, weekly thereafter, and on the day of each scheduled sacrifice. Neurobehavioral observations were performed weekly; hand-held and open-field expanded clinical observations were done pre-study and during Weeks 4, 8, and 13; elicited behavior observations were done prestudy and during Wk 13; and motor activity data were collected pre-study and during Wk 13. Ophthalmic examinations were done prior to treatment and during Wk 13. Body weights were collected twice prior to treatment, on Day 1, and weekly thereafter. Food consumption data were measured weekly. Blood and urine samples for hematology, coagulation, clinical chemistry, urinalysis, and urine chemistry were collected at each scheduled sacrifice (on Day 92 and Day 120).

Furthermore, vaginal cytology and sperm analysis were performed.

There were no test substance-related effects on ophthalmic observations; effects on neurobehavioral assessment tests; effects on body weights or body weight changes; effects on food consumption; or effects on vaginal cytology. Test substance had no effect on clinical pathology test results.

The mean percent sperm motility, caudal epididymal sperm count, and sperm morphology were not affected by treatment.No biologically meaningful differences were observed between the study groups.

 

Based on the results of the study, the No Observed Adverse Effect Level (NOAEL) following oral gavage administration of N,N-bis (2-hydroxyethyl)-PPD Sulf to rats at doses of 0, 1, 4, or 20 mg/kg/day for 91 d was determined to be 20 mg/kg/day. No toxicity on reproductive apparatus or fertily was observed.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no adverse effect observed

Effects on developmental toxicity

Description of key information

The maternal and developmental toxicity potential of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate was determined following the OECD Guideline 414 (Prenatal Developmental Toxicity Study).

Administration of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate by oral gavage to mated female Crl:CD (SD)IGS BR VAF/Plus rats during gestation Day 6-20 at dose levels of 0, 5, 20 and 50 mg /kg bw/day revealed a NOAEL of 5 mg/kg bw/day for maternal toxicity and 50 mg/kg bw/day for developmental toxicity.  

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 11, 2004 to June 04, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed method comparable to guideline, GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Remarks:

according to U.S. FDA, OECD and JMAFF Principles of GLP

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD)IGS BR VAF/Plus

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: 68 d
- Weight at study initiation: 219 - 247 g (weight of females at study assignment)
- Fasting period before study: No
- Housing: individually in stainless steel, wire-bottomed cages, except during the cohabitation period. The study rooms were maintained under conditions of positive airflow relative other places.
- All cage sizes and housing conditions were in compliance with the "Guide for the Care and Use of Laboratory Animals". Cage pan liners were changed at least three times weekly. Cages were changed approximately every other week.
- Diet: Certified Rodent Diet # 5002 (PMI Nutrition International, St. Louis, Missouri, USA) in individual feeders; ad libitum. Diet was routinely analysed by the feed supplier. No contaminants at levels exceeding the maximum concentration limits for certified feed or deviations from expected nutritional requirements were detected by these analyses.
- Water: Local water (purified by reverse osmosis) provided from an automatic watering access system; ad libitum. The processed water was analyzed twice annually for possible chemical contamination (Lancaster Laboratories, Lancaster, Pennsylvania, USA) and monthly for possible bacterial contamination (QC Laboratories, Southampton, Pennsylvania, USA).
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature: 69.0-80°F (20.6-26.7°C).
- Relative humidity: 39.0-57.2%.
- Air changes: A minimum of 10 air changes/h with 100% fresh air that had been passed through 99.97% HEPA filters.
- Photoperiod: An automatically controlled 12 h fluorescent light/12 h dark cycle/d.

IN-LIFE DATES: From: May 17, 2004 To: June 04, 2004

Route of administration:

oral: gavage

Vehicle:

other: aqueous 0.2% w/v erythorbic acid (prepared using erythorbic acid (D-araboascorbic acid; isoascorbic acid), a white, crystalline powder, and reverse osmosis membrane processed deionized water (R.O. deionized water))

Details on exposure:

PREPARATION OF DOSING FORMULATIONS (SOLUTIONS): Appropriate (weighed) quantity of test substance was mixed thoroughly with vehicle using magnetic stirer until the test substance was completely dissolved.
- Rate of preparation of dosing solutions: Weekly
- Storage condition of dosing solutions: Refrigerated (4-8°C)

VEHICLE
- Justification for use and choice of vehicle: Not reported
- Concentration in vehicle: aqueous 0, 0.5, 2 and 5 mg/mL
- Amount of vehicle: 10 mL/kg bw (adjusted daily on the basis of the individual body weights recorded immediately before administration of the test substance).
- Lot/batch no. ( erythrobic acid): Spectrum Chemical MFG. CORP; Lot# SW0767

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Homogeneity and concentration (all levels) analyses were performed by HPLC method (Covance Method Procedure MA-PG61-2050) in Covance Laboratories Inc. Wisconsin 53704 USA.
- For Homogeneity analysis, quadruplicate samples were taken from the top, middle and bottom of the high dose concentration (5.0 mg/mL) from the first preparation used on this study to establish homogeneity of the high dose formulation. Two samples from each quadruplicate were shipped for analysis.
- For concentration analysis, quadruplicate samples were taken from the middle of all dose formulations on all preparations performed on the study. Two samples from each quadruplicate set were shipped for analysis. For the first preparation on study, the mean of the homogeneity sample concentration values served as the concentration value for the high dose formulation.
- The mean concentration results for all samples analyzed varied from 95.0% to 104% of the respective theoretical value. For the homogeneity analyses, the overall relative standard deviation from all sampling locations for each respective concentration was within 3%.
- Stability of the dose formulations was established by Covance study# 6114-461 and this information was provided to the Study Director. No stability determination was performed during the conduct of this study.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1M:1F
- Length of cohabitation: 4 d
- Verification of same strain and source of both sexes: Male and female rats were of same strain and were obtained from same source.
- Proof of pregnancy: Mating was evaluated daily during the cohabitation period and confirmed by observation of spermatozoa in a smear of the vaginal contents and/or a copulatory plug observed in situ. The Day mating was confirmed was designated as Day 0 of (presumed) gestation.

Duration of treatment / exposure:

Gestation Day 6 through Day 20

Frequency of treatment:

Daily (at approx. the same time each day)

Duration of test:

21 d

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 mated females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were selected on the basis of a range-finding study (Argus Protocol 916-061P). The highest dosage level selected was anticipated to induce some overt developmental and/or maternal toxicity (clinical signs or a decrease in body weight) but not death or severe suffering. The intermediate dose level was selected to produce minimal observable toxicity. The lowest dosage level was selected to not produce any evidence of either maternal or developmental toxicity. A descending sequence of dose levels was selected with the purpose of demonstrating any dosage-related response and to establish a no-observed-adverse-effect level (NOAEL).
Available additional information on metabolism and kinetics of the test substance or related materials was also considered.
- Rationale for animal assignment: Rats were assigned to individual housing on the basis of computer-generated random units. Healthy, mated female rats were assigned to four dosage groups (1 control and 3 treatment), 25/group, by using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on Gestation Day 0.

Maternal examinations:

MORTALITY: Yes
- Time schedule: At least twice each day

DETAILED CLINICAL OBSERVATIONS: Yes, animals were examined for clinical observations, general appearance, abortions and premature deliveries.
- Time schedule: Weekly during the acclimation period and on gestation Day 0, thereafter daily before and after dosage and on the day of sacrifice.
Post dosage observations were recorded at approx. hourly intervals for the first 4 h and at the end of the normal working day on the first three days of dosage administration. On subsequent days, post dosage observations were recorded approx. 2 h (±30 min) after administration.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during the acclimation period, on Gestation Day 0 and daily during the dosage and post dosage periods.

FOOD CONSUMPTION: Yes, feed consumption values were recorded on Gestation Day 6, 9, 12, 15, 18, and 20

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21, all surviving animals were sacrificed by CO2 asphyxiation and caesarean sectioned.
- Organs examined: Gross necropsy of the thoracic, abdominal and pelvic viscera was performed.
- Gross lesions were retained in neutral buffered 10% formalin for possible future evaluation. A table of random units was used to select one control group rat from which all tissues examined at necropsy were retained, in order to provide comparative tissues for any possible histopathological evaluations of gross lesions.
- To minimize bias, Caesarean-sectioning and subsequent fetal observations were conducted without knowledge of dosage group.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number and distribution of corpora lutea: Yes
- Number and distribution of implantations: Yes
- Number of early resorptions: Yes (early resorption was defined as one in which organogenesis was not grossly evident).
- Number of late resorptions: Yes (late resorption was defined as one in which the occurrence of organogenesis was grossly evident).
- Number of live and dead fetuses: Yes (A live fetus was defined as a term fetus that responded to stimuli. Nonresponding term fetuses were considered to be dead (there were no dead fetuses). Dead fetuses and late resorptions were differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetus was a late resorption).
- Uteri of apparently nonpregnant rats were stained with 10% ammonium sulfide to confirm the absence of implantation sites.
- Other: Placentae were examined for size, color and shape.

Fetal examinations:

- External examinations: Yes: all per litter. Each fetus was removed from the uterus, placed in an individual container and individually identified with a tag noting the study number, litter number, uterine distribution and fixative.
- Each fetus was subsequently weighed and examined for sex and gross external alterations. Live fetuses were sacrificed by an intraperitoneal injection of EUTHASOL (Diamond Animal Health, Inc., Des Moines, Iowa, USA).
- Soft tissue examinations: Yes: half per litter. Totals of 167, 175, 129 and 157 fetuses were examined for soft tissue alterations in the four respective dosage groups following a variation of the microdissection technique of Staples.
- Skeletal examinations: Yes: half per litter. Totals of 177, 189, 137 and 170 fetuses were examined for skeletal alterations and fetal ossification site averages, in the four respective dosage groups. Fetuses were eviscerated, cleared, stained with alzarin red and examined for skeletal alterations. The fetuses were initially fixed in alcohol. Skeletal preparations were retained in glycerin, with thymol added as a preservative.
- Head examinations: Yes: half per litter. The heads of the fetuses undergoing soft tissue examinations were fixed in Bouin's solution and subsequently examined by free-hand sectioning; head sections were retained in alcohol.

Statistics:

- All data were tabulated, summarized and/or statistically analyzed using the Argus Automated Data Collection and Management System, the Vivarium Temperature and Relative Humidity Monitoring System, Microsoft Excel (part of Microsoft Office 97/2000/XP), Quattro Pro 8 and/or The SAS System (version 6.12). Averages and percentages were calculated. Litter values were used where appropriate. Statistically significant probabilities were reported as either p<=0.05 or p<=0.01.
- Clinical observations and other proportional data were analyzed for the Variance Test for Homogeneity of the Binomial Distribution. Continuous data (e.g., maternal body weights, body weight changes, feed consumption values, organ weights and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and fetal anomaly data) were analyzed using Bartlett's Test of Homogeneity of Variances and the Analysis of Variance, where appropriate [i.e., Bartlett's Test was not significant (P>0.001)].
- If the Analysis of Variance was significant (p<=0.05), Dunnett's Test was used to identify the statistical significance of the individual groups. If the analysis of Variance was not appropriate [i.e., Bartlett's Test was significant (p<=0.001)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p<=0.05), Dunn's Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher's Exact Test was used to analyze the data. Count data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.

Historical control data:

- Summary of following historical control data has been provided in the report:

Summary of reproductive indices, summary of maternal necropsy observations Crl:CD(SD)IGS BR rats - Day 21 C-section, summary of fetal gross external alterations Crl:CD(SD)IGS BR rats - Day 21 C-section, summary of fetal soft tissue alterations Crl:CD(SD)IGS BR rats - Day 21 C-section, summary of fetal skeletal alterations Crl:CD(SD)IGS BR rats - Day 21 C-section, summary of fetal ossification sites skeletal averages Crl:CD(SD)IGS BR rats - Day 21 C-section.

Details are provided in the study report.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical observations related to the test substance occurred. Variations like localized alopecia in limb(s) or head, chromorhinorrhea, urine-stained abdominal fur, rales, mass on the nose, incisor(s) missing/broken and scant feces were observed. However, these findings were not considered to be treatment related as they were sporadic incidences or were common in species and strain of the test animal.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One dam in the high dose group was found dead on Day 18 of gestation. No specific cause of death was determined and was therefore not considered to be treatment related.
- All other dams survived to scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weight gains were reduced in the mid dose group and significantly reduced (p≤0.05) in the high dose group animals during the entire dosage period when compared to vehicle control group value.
Body weight gains in the low, mid and high dose groups were 107.1%, 92.6% and 90.5% of the vehicle control group value respectively.
During the dosing period significant reductions in body weight gains occurred during gestation Day 6 to 9 for high and mid dose groups. Body weight gains in the mid and high dose groups were 76.1% and 65.7% of control value respectively.
Body weights were significantly reduced on gestation Day 8 and 11 to 21 in the mid dose group (p≤0.05 or p≤0.01) and on gestation Day 8, 18, 19 and 21 in the high dose group (p≤0.05), as compared to the vehicle control group. Gravid uterine weights were reduced in the mid and high dose groups (91.3% and 91.0%, respectively, of the control value), as compared to the vehicle control value. These reductions were not dosage dependent and reflected a slightly smaller litter size that occurred spontaneously in the mid and high dose groups.
Corrected maternal body weights (gestation Day 21 body weight minus the gravid uterine weight) and corrected body weight changes (calculated as gestation Day 6 to 21 and 0 to 21) were comparable among the groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Corresponding to reductions in body weight gain, absolute (g/day) and relative (g/kg/day) feed consumption values were significantly reduced in the mid dose group (p≤0.05 or p≤0.01) and high dose group (p≤0.05) for the dosage interval of gestation Day 6 to 9 (92.2% and 92.5%, respectively, of the vehicle control group absolute value).
Absolute feed consumption values in the low, mid, and high dose groups were 101.1%, 96.6% and 97.0%, respectively, of the vehicle control group values forthe entire dosage period (calculated as gestation Day 6 to 21).
Absolute and relative feed consumption values were unaffected in the low dose group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment related gross lesions were observed in necropsy. Isolated occurrence of urinary tract lesions was noted in one rat; this is a common finding in this species and strain and was considered unrelated to the treatment.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

CAESAREAN-SECTIONING AND LITTER OBSERVATIONS: Pregnancy occurred in 24 (96.0%), 24 (96.0%), 20 (80.0%) and 25 (100%) rats in the control, low, mid and high dose groups respectively. One dam in the control group delivered its litter on gestation Day 21 and was sacrificed. This dam was given 15 dosages of the vehicle, and had no adverse clinical observations.
- No treatment related effects were noted in caesarean-sectioning or litter observations. The litter averages for corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, fetal body weights, percent resorbed conceptuses and percent live male fetuses were comparable among the four dosage groups and did not significantly differ.
There were no dead fetuses and no litters with all conceptuses resorbed; all placentae appeared normal. All values were within the ranges observed historically at the testing facility.

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: effects obserevd in the 20 and 50 mg/kg/day dosage groups

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

effects observed, non-treatment-related

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Excluding fetuses from the dam that was found dead in the high dose group and fetuses from the dam that delivered on gestation Day 21 in the control group, fetal evaluations were based on 344, 364, 266 and 327 live, caesarean-delivered fetuses from 23, 24, 20 and 24 litters in the control, low, mid and high dose groups, respectively.
- Summary of fetal alterations: In four respective dose groups (control, low, mid and high) fetal alteration were observed in 3 (13.0%), 8 (33.3%; significant at p<=0.05), 10 (50%; significant at p<=0.01) and 5 (20.8%) litters respectively.
- The numbers of fetuses with any alteration observed were 3 (0.9%), 11 (3.0%; significant at p≤0.01), 11 (4.1%; significant at p≤0.01) and 6 (1.8%), in control, low, mid and high dose group respectively.
- The percentages of fetuses with any alteration per litter were 0.8, 3.3, 5.4 (significant at p≤0.01) and 1.8 for the four dosage groups, respectively.
- The significant increases (p≤0.05 to p≤0.01) in the number of litters with fetuses with alterations, the number of fetuses with alterations and the percentage of fetuses with alterations per litter were not considered related to the test substance, as the increases were not dosage dependent and) the increases reflected low values in the control group.
- With the exception of wavy ribs that occurred in three (significant at p≤0.01) fetuses in two high dose group litters, there were no dosage-dependent significant differences in the litter or fetal incidences of any gross external, soft tissues or skeletal alterations. The incidence of wavy ribs was not considered related to the test substance because it was within the historical control range for this test facility and the more relevant litter incidence was not significantly increased. There was a significant (p≤0.05) decrease in the number of ossified hindlimb phalanges in the high dose group; however, this was not considered toxicologically important since there was no other adverse effect on fetal weights or fetal alterations.

- There was a significant (p≤0.05) decrease in the number of ossified hindlimb phalanges in the 50 mg/kg/day dosage group; however, this was not considered toxicologically important since there was no other adverse effect on fetal weights or fetal alterations. No other gross external, soft tissue or skeletal fetal alterations (malformations or variations) were attributable to treatment with the test substance.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no developmental toxicity observed up to the highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Administration of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate by oral gavage to mated female Crl:CD (SD)IGS BR VAF/Plus rats during gestation Day 6-20 at dose levels of 0, 5, 20 and 50 mg /kg bw/day revealed a NOAEL of 5 mg/kg bw/day for maternal toxicity and 50 mg/kg bw/day for developmental toxicity.

Executive summary:

The maternal and developmental toxicity potential of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate was determined following the OECD Guideline 414 (Prenatal Developmental Toxicity Study).

The purpose of this study was to evaluate the developmental toxicity (embryo-fetal toxicity and teratogenic potential) of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate when administered to female Crl:CD (SD)IGS BR VAF/Plus rats (presumed pregnant) by oral gavage at dose levels of 0, 5, 20 and 50 mg /kg bw/day for a period of gestation Day 6 through 20.

One hundred female Crl:CD (SD)IGS BR VAF/Plus rats of 68 d age (Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA) weighing 219 - 247 g were housed individually in stainless steel, wire-bottomed cages. The animals were maintained under standard laboratory conditions (temperature: 20.6-26.7°C, humidity: 39.0-57.2%, minimum of 10 air changes/h, 12 h light/12 h dark cycle per d) and fed on Certified Rodent Diet #5002; ad libitum. The animals were acclimatized for 6 d prior to mating.

 

After 6 d of acclimation, 140 virgin female rats were placed into cohabitation with 140 breeder male rats, one male rat per female rat for a 4 d cohabitation period. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were confirmed pregnant (The day pregnancy was confirmed was designated as gestation Day 0). Post-coitum, mated females were randomly divided into 3 different treatment groups consisting of 25 animals/dose group. A similar sized group of animals that received 0.2% w/v erythorbic acid in R.O. deionized water (vehicle) served as control. Solutions of the test substance and/or the vehicle were administered orally (gavage) once daily from Days 6 through 20 of presumed gestation. The dosage volume was 10 mL/kg, adjusted daily on the basis of the individual body weights recorded immediately before administration of the test substance.

 

Viabilities, clinical observations, body weights and feed consumption values were recorded. All surviving rats were sacrificed on gestation Day 21. The gravid uterus was excised, weighed and subsequently examined for the number and distribution of corpora lutea, implantation sites and uterine contents. A gross necropsy was performed. Fetuses were weighed and examined for gross external, soft tissue and skeletal alterations and sex. To minimize bias, Caesarean-sectioning and subsequent fetal observations were conducted without knowledge of dosage group.

One dam in the high dose group was found dead on gestation Day 18. No other mortality or any clinical necropsy observations related to the test substance occurred. Maternal body weight gains were reduced in the mid dose group and significantly reduced in the high dose group during the entire dosage period (calculated as gestation Day 6 -21). The gravid uterine weights were reduced in the mid and high dose groups, as compared to the control group value. Although not always dosage dependent, these reductions reflected a slightly smaller litter size that occurred spontaneously in the mid and high dose groups (13.3 and 13.6, respectively) compared to the 0 (Vehicle) and low dose groups (15.0 and 15.2, respectively). Corrected maternal body weights (gestation Day 21 body weight minus the gravid uterine weight) and corrected body weight changes were comparable among the groups. Corresponding to reductions in body weight gain, absolute (g/day) and relative feed consumption values (g/kg/day) were significantly reduced in the mid and high dose groups for the dosage interval of gestation Day 6 -9 (92.2% and 92.5%, respectively, of the control group absolute value).

No treatment related effects were noted in caesarean-sectioning or litter observations. The litter averages for corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, fetal body weights, percent resorbed conceptuses and percent live male fetuses were comparable among the four dosage groups and did not differ significantly.

The significant increases (p<=0.05 to p<=0.01) in the number of litters with fetuses with alterations, the number of fetuses with alterations and the percentage of fetuses with alterations per litter were not considered related to the test substance as the increases were not dosage dependent and the increases reflected low values in the control group. There was a significant (p≤0.05) decrease in the number of ossified hindlimb phalanges in the 50 mg/kg/day dosage group; however, this was not considered toxicologically important since there was no other adverse effect on fetal weights or fetal alterations. No other gross external, soft tissue or skeletal fetal alterations (malformations or variations) were attributable to treatment with the test substance. 

Based on the above observations, the maternal no-observable-adverse-effect-level (NOAEL) was 5 mg/kg/day. Dosages of 20 and 50 mg/kg/day produced reductions in body weight and feed consumption. The developmental NOAEL was determined by the study authors to be 50 mg/kg/day. No developmental toxicity was produced at dosages as high as 50 mg/kg/day, the highest dose tested.

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

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Mar. 22, 2004 to Apr. 14, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Range finding study, well documented, comparable to scientific standards/principles, GLP.

Qualifier:

no guideline available

Principles of method if other than guideline:

This dose range-finding prenatal developmental toxicity study was performed to establish suitable dose levels of test substance for definitive developmental toxicity study. Eight mated female rats per group received test solution/vehicle orally, at dose levels of 0, 5, 10, 20 or 40 mg/kg bw/day daily from Gestation Day (GD) 6-20. All females were sacrificed on GD 21 and caesarean section examination was performed.

GLP compliance:

yes

Remarks:

according to U.S. FDA and OECD Principles of GLP

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD)IGS BR VAF/Plus

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: 62 d
- Weight at study initiation: 219 - 242 g (females at study assignment)
- Fasting period before study: No
- Housing: Individually housed in stainless steel, wire-bottomed cages, except during the cohabitation period. The study rooms were maintained under conditions of positive airflow relative other places.
- All cage sizes and housing conditions were in compliance with the "Guide for the Care and Use of Laboratory Animals". Cage pan liners were changed at least three times weekly. Cages were changed approximately every other week.
- Diet: Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri, USA) in individual feeders; ad libitum
- Diet was routinely analysed by the feed supplier. No contaminants at levels exceeding the maximum concentration limits for certified feed or deviations from expected nutritional requirements were detected by these analyses.
- Water: Local water (purified by reverse osmosis) provided from an automatic watering access system; ad libitum.
The processed water was analyzed twice annually for possible chemical contamination (Lancaster Laboratories, Lancaster, Pennsylvania, USA) and monthly for possible bacterial contamination (QC Laboratories, Southampton, Pennsylvania, USA).
- Acclimation period: 4 d

ENVIRONMENTAL CONDITIONS
- Temperature: 66-72.4°F (19-22.4°C).
- Relative humidity: 28.2-55.4%.
- Air changes: A minimum of 10 air changes/h with 100% fresh air that had been passed through 99.97% HEPA filters.
- Photoperiod: Automatically controlled 12 h fluorescent light/12 h dark cycle per d

IN-LIFE DATES: From: Mar. 28, 2004 To: Apr. 14, 2004

Route of administration:

oral: gavage

Vehicle:

other: Aqueous 0.2% w/v erythrobic acid (prepared using erythorbic acid (D-araboascorbic acid; isoascorbic acid), a white, crystalline powder, and reverse osmosis membrane processed deionized water)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Appropriate (weighed) quantity of test substance was mixed thoroughly with vehicle using magnetic stirrer until the test substance was completely dissolved.
- Rate of preparation of dosing solutions: Weekly
- Storage condition of dosing solutions: Refrigerated (4-8°C)

VEHICLE
- Justification for use and choice of vehicle: Not reported
- Concentration in vehicle: 0, 0.5, 1, 2 and 4 mg/mL
- Amount of vehicle: 10 mL/kg bw (adjusted daily on the basis of the individual body weights recorded immediately before administration of the test substance).
- Lot/batch no.(erythrobic acid): Spectrum Chemical MFG. CORP; Lot# SW0767

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Homogeneity and concentration (all levels) analyses were performed by HPLC method (Covance Method Procedure MA-PG61-2050) in Covance Laboratories Inc. Wisconsin 53704 USA.
- For Homogeneity analysis, quadruplicate samples were taken from the top, middle and bottom of the lowest and the highest concentrations (0.5 and 4.0 mg/mL) from the first day of preparation. Two samples from each quadruplicate were shipped for analysis.
- For concentration analysis, quadruplicate samples were taken from the middle of the control and intermediate concentrations (0, 1.0 and 2.0 mg/mL) from the first day of preparation and from the middle of all concentrations for all subsequent preparations. Two samples from each quadruplicate set were shipped for analysis. For the first preparation on study, the mean of the homogeneity sample concentration values served as the concentration value for the lowest and highest concentrations (0.5 and 4.0 mg/mL, respectively).
- The mean concentration results for all samples analyzed varied from 96.3% to 103% of the respective theoretical value. For the homogeneity analyses, the overall relative standard deviation from all sampling locations for each respective concentration was within 2%.

Details on mating procedure:

- Impregnation procedure: Cohoused (in the male rat's cage)
- M/F ratio per cage: 1 M/1 F
- Length of cohabitation: 4 d
- Verification of same strain and source of both sexes: Male and female rats were of same strain and were obtained from same source.
- Proof of pregnancy: Mating was evaluated daily during the cohabitation period and confirmed by observation of spermatozoa in a smear of the vaginal contents and/or a copulatory plug observed in situ. The Day mating was confirmed was designated as Day 0 of (presumed) gestation.

Duration of treatment / exposure:

Gestation Day 6 through Day 20

Frequency of treatment:

Daily (at approx. the same time each day)

Duration of test:

21 d

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

40 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

8 mated females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were selected by the Sponsor on the basis of existing data on this test substance. The highest dose level was selected to induce some overt developmental and/or maternal toxicity (clinical signs or a decrease in body weight) but not death or severe suffering. At least one intermediate dose level was selected to produce minimal observable toxicity.
The lowest dose level was chosen so that it would not produce any evidence of either maternal or developmental toxicity. A descending sequence of dosage levels was selected with the purpose of demonstrating any dose-related response and to establish a no-observed-adverse-effect level (NOAEL). Available additional information on metabolism and kinetics of the test substance and related materials was considered.
- Rationale for animal assignment: Rats were assigned to individual housing on the basis of computer-generated random units. Healthy, mated female rats were assigned to five dosage groups (1 control and 4 treatment), 8/group, by using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on GD 0.
- Assignment of animals: The animals were assigned into following groups in the study report:
Group I (Negative control): 0 mg/kg bw/day
Group II (Low dose): 5 mg/kg bw/day
Group III (Lower mid dose): 10 mg/kg bw/day
Group IV (Higher mid dose): 20 mg/kg bw/day
Group V (High dose): 40 mg/kg bw/day

Maternal examinations:

MORTALITY: Yes
- Time schedule: At least twice each day

DETAILED CLINICAL OBSERVATIONS: Yes, animals were examined for clinical observations, general appearance, abortions and premature deliveries.
- Time schedule: Weekly during the acclimation period and on GD (Gestation Day) 0, thereafter daily before and after dosage and on the day of sacrifice.
- Post dosage observations were recorded at approx. hourly intervals for the first 4 h and at the end of the normal working day on the first 3 d of dosage administration. From Day 4 onwards, clinical observations were recorded approximately 60 ± 10 min after dosage administration.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during the acclimation period, on gestation Day 0 and daily during the dosage and post dosage periods.

FOOD CONSUMPTION: Yes (recorded on gestation Day 0, 6, 9, 12, 15, 18, 20 and 21.)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day # 21, all surviving animals were sacrificed by CO2 asphyxiation and caesarean sectioned.
- Organs examined: Gross necropsy of the thoracic, abdominal and pelvic viscera was performed.
- A table of random units was used to select one control group rat from which all tissues examined at necropsy were retained, in order to provide comparative tissues for any possible histopathological evaluations of gross lesions.
- To minimize bias, Caesarean-sectioning and subsequent fetal observations were conducted without knowledge of dose group.

Ovaries and uterine content:

- The ovaries and uterine content were examined after termination: Yes

Examinations included:
- Gravid uterus weight: Yes
- Number and distribution of corpora lutea: Yes
- Number and distribution of implantations: Yes
- Number of early resorptions: Yes (early resorption was defined as one in which organogenesis was not grossly evident).
- Number of late resorptions: Yes (late resorption was defined as one in which the occurrence of organogenesis was grossly evident).
- Number of live and dead fetuses: Yes (A live fetus was defined as a term fetus that responded to stimuli. Non-responding term fetuses were considered to be dead (there were no dead fetuses). Dead fetuses and late resorptions were differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetus was a late resorption).
- Uteri of apparently nonpregnant rats were stained with 10% ammonium sulfide to confirm the absence of implantation sites.
- Other: Placentae were examined for size, color and shape.

Fetal examinations:

- External examinations: Yes: all per litter. Fetuses were examined for sex and gross external alterations. Late resorptions were examined for gross external alterations to the extent possible. The body weight of each fetus was recorded. Only body weights of live fetuses were used to determine litter fetal body weight averages.
Live fetuses were sacrificed by an intraperitoneal injection of sodium pentobarbital. Fetuses with gross external alterations were fixed in Bouin's solution; all other fetuses were discarded.
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:

- All data were tabulated, summarized and/or statistically analyzed using the Argus Automated Data Collection and Management System, the Vivarium Temperature and Relative Humidity Monitoring System, Microsoft Excel (part of Microsoft Office 97/2000/XP), Quattro Pro 8 and/or The SAS System (version 6.12). Averages and percentages were calculated. Litter values were used where appropriate.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All rats survived to scheduled sacrifice. The only clinical observation considered to be related to the test substance was a pink substance in each cage pan, which was observed on single days in two or three rats in the group IV and V (GD 16, 18 or 20). Variations like localized alopecia (limbs and/or back) and soft or liquid feces were observed. However, these findings were not considered to be treatment related as they were sporadic incidences or incidences were not dosage dependent.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Maternal body weights were comparable among the five dosage groups through GD 21. Gravid uterine weights and corrected maternal body weights (GD 21 body weight minus the gravid uterine weight) were similar among the dosage groups.

- Body weight gains were slightly reduced in group III, IV and V, in comparison with the control group value during GD 6 to 9, following the initiation of the treatment but were comparable among the dosage groups for all other tabulated intervals, including the dosage and gestation periods.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross lesions were revealed by necropsy examination.

Other effects:

no effects observed

Description (incidence and severity):

Gravid uterine weights and corrected maternal body weights (GD 21 body weight minus the gravid uterine weight) were similar among the dosage groups. No statististically significant differences in body weight gains were noted.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

CAESAREAN-SECTION OBSERVATION: Pregnancy occurred in 6 to 8 rats in each dose group. Caesarean-sectioning observations were based on 7 (87.5%), 8 (100%), 8 (100%), (75.0%), and 7 (87.5%) pregnant rats with one or more live fetuses in Groups I through V, respectively.
No Caesarean-sectioning or litter parameters were affected by treatment. The litter averages for corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, fetal body weights, percent resorbed conceptuses, and percent live male fetuses were comparable among the five dosage groups. No dam had a litter consisting of only resorbed conceptuses, and there were no dead fetuses.

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

External malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Totals of 92, 106, 111, 85 and 99 fetuses in the five respective dose groups were examined externally. One fetus in the highest dose group (V) had anumbilical hernia. No other alterations were observed at fetal gross external examination.

Dose descriptor:

NOAEL

Effect level:

>= 40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no developmental toxicity observed up to the highest dose tested

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Administration of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate by oral gavage to mated female Crl:CD (SD)IGS BR VAF/Plus rats from gestation Day 6-20 at dose levels of 0, 5, 10, 20 and 40 mg /kg bw/day revealed a NOAEL of 20 mg/kg bw/day (maternal toxicity) and 40 mg/kg bw/day (developmental toxicity).
Based on the results, dose levels of 0, 5, 20 and 50 mg/kg bw/day were considered appropriate for a subsequent main study.

Executive summary:

The purpose of this dose range-finding prenatal developmental toxicity study was to assess the effects of N,N-bis

(2-hydroxyethyl)-p-phenylenediamine sulfate on pregnant female Crl:CD (SD)IGS BR VAF/Plus rats and embryo-fetal development when

administered by oral gavage at dose level of 0, 5, 10, 20 and 40 mg /kg bw/day from Gestation Day (GD) 6 through 20 and also to establish suitable dose levels of test substance for subsequent main study.

Forty female Crl:CD (SD) IGS BR VAF/Plus rats of 62 d age (Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA) weighing 219-242 g were housed individually in stainless steel, wire-bottomed cages. The animals were maintained under standard laboratory conditions (temperature: 19-22.4°C, relative humidity: 28.2-55.4%, minimum of 10 air changes/h, 12 h light/12 h dark cycle per d) and fed on Certified Rodent Diet # 5002; ad libitum. The animals were acclimatized for 4 d prior to mating.

 

After 4 d of acclimation, 40 virgin female rats were placed into cohabitation with 40 breeder male rats (one male rat per female rat) for a 4 d cohabitation period. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were confirmed pregnant (The day of confirmation of pregnancy was referred to as gestation Day 0). Post-coitum, mated females were randomly divided into 4 different treatment groups consisting of 8 mated females/dose group. A similar sized group of animals that received 0.2% w/v erythorbic acid in R.O. deionized water (vehicle) served as control. Solutions of the test substance and/or the vehicle were administered orally (gavage) once daily from Days 6 through 20 of presumed gestation. The dosage volume was 10 mL/kg, adjusted daily on the basis of the individual body weights recorded immediately before administration of the test substance.

 

Viabilities, clinical observations, body weights and food consumption values were recorded. All rats were sacrificed on GD 21. The gravid uterus was excised, weighed and subsequently examined for the number and distribution of corpora lutea, implantation sites and uterine contents. A gross necropsy was performed. Fetuses were weighed and examined for gross external alterations and sex.

The only clinical observation considered to be related to the test substance was a pink substance in cage pan, which was observed on single days in two or three rats in the group IV and V(GD 16, 18, or 20). All rats survived to scheduled sacrifice. No gross lesions were revealed by necropsy examination. Body weight gains were slightly reduced on GD 6 to 9 in the group III, IV and V, but were comparable among the dosage groups for all other tabulated intervals, including the dosage and gestation periods. Corrected maternal body weight gains were reduced in the highest dose group (V) for the dosage and gestation periods, in comparison with the respective control group values. Absolute and relative feed consumption values were comparable among the five dosage groups for the treatment and gestation periods. No caesarean-sectioning or litter parameters were affected by treatment at all concentrations tested. One fetus in the 40 mg/kg/day dosage group had an umbilical hernia.

Based on reduced corrected maternal body weight gains seen in the high dose group, the NOAEL for maternal toxicity was determined to be 20 mg/kg bw/day. The NOAEL for developmental toxicity was determined to be 40 mg/kg bw/day.

Based on the findings, dose levels of 0, 5, 20 and 50 mg/kg bw/day was considered appropriate for a subsequent main study.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Four studies were available in order to assess the potential teratogenicity effect of the registered substances. They were performed according to OECD 414 method :

-York R.G., 2005, Klimisch 1, Dose range finding study :

This dose range-finding prenatal developmental toxicity study was to assess the effects of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate on pregnant female Crl:CD (SD)IGS BR VAF/Plus rats and embryo-fetal development when administered by oral gavage at dose level of 0, 5, 10, 20 and 40 mg /kg bw/day from Gestation Day (GD) 6 through 20 and also to establish suitable dose levels of test substance for subsequent main study.

Forty female Crl:CD (SD) IGS BR VAF/Plus rats of 62 d age were used. Solutions of the test substance and/or the vehicle were administered orally (gavage) once daily from Days 6 through 20 of presumed gestation. The dosage volume was 10 mL/kg, adjusted daily on the basis of the individual body weights recorded immediately before administration of the test substance. Viabilities, clinical observations, body weights and food consumption values were recorded. All rats were sacrificed on GD 21. The gravid uterus was excised, weighed and subsequently examined for the number and distribution of corpora lutea, implantation sites and uterine contents. A gross necropsy was performed. Fetuses were weighed and examined for gross external alterations and sex.

The only clinical observation considered to be related to the test substance was a pink substance in cage pan, which was observed on single days in two or three rats in the group IV and V(GD 16, 18, or 20). All rats survived to scheduled sacrifice. Body weight gains were slightly reduced on GD 6 to 9 in the group III, IV and V, but were comparable among the dosage groups. Corrected maternal body weight gains were reduced in the highest dose group (V) for the dosage and gestation periods, in comparison with the respective control group values.Absolute and relative feed consumption values were comparable among the five dosage groups for the treatment and gestation periods. No caesarean-sectioning or litter parameters were affected by treatment at all concentrations tested. One fetus in the 40 mg/kg/day dosage group had an umbilical hernia. Based on reduced corrected maternal body weight gains seen in the high dose group, the NOAEL for maternal toxicity was determined to be 20 mg/kg bw/day. The NOAEL for developmental toxicity was determined to be 40 mg/kg bw/day.

Based on the findings, dose levels of 0, 5, 20 and 50 mg/kg bw/day was considered appropriate for a subsequent main study.

-York, R.G., 2005, Klimisch 1, OECD 414 guideline, Main study :

The purpose of this study was to evaluate the developmental toxicity (embryo-fetal toxicity and teratogenic potential) of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate when administered to female Crl:CD (SD)IGS BR VAF/Plus rats (presumed pregnant) by oral gavage at dose levels of 0, 5, 20 and 50 mg /kg bw/day for a period of gestation Day 6 through 20.

One hundred female Crl:CD (SD)IGS BR VAF/Plus rats of 68 d age. After 6 d of acclimation, 140 virgin female rats were placed into cohabitation with 140 breeder male rats, one male rat per female rat for a 4 d cohabitation period. Post-coitum, mated females were randomly divided into 3 different treatment groups consisting of 25 animals/dose group Solutions of the test substance and/or the vehicle were administered orally (gavage) once daily from Days 6 through 20 of presumed gestation. The dosage volume was 10 mL/kg, adjusted daily on the basis of the individual body weights.

Viabilities, clinical observations, body weights and feed consumption values were recorded. All surviving rats were sacrificed on gestation Day 21. The gravid uterus was excised, weighed and subsequently examined. A gross necropsy was performed. Fetuses were weighed and examined for gross external, soft tissue and skeletal alterations and sex.

One dam in the high dose group was found dead on gestation Day 18. No other mortality or any clinical necropsy observations related to the test substance occurred. Maternal body weight gains were reduced in the mid dose group and significantly reduced in the high dose group during the entire dosage period (calculated as gestation Day 6 -21). The gravid uterine weights were reduced in the mid and high dose groups, as compared to the control group value. Although not always dosage dependent, these reductions reflected a slightly smaller litter size that occurred spontaneously in the mid and high dose groups (13.3 and 13.6, respectively) compared to the 0 (Vehicle) and low dose groups (15.0 and 15.2, respectively, absolute (g/day) and relative feed consumption values (g/kg/day) were significantly reduced in the mid and high dose groups for the dosage interval of gestation Day 6 -9.

No treatment related effects were noted in caesarean-sectioning or litter observations. The litter averages for corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, fetal body weights, percent resorbed conceptuses and percent live male fetuses were comparable among the four dosage groups and did not differ significantly.

Based on the above observations, the maternal no-observable-adverse-effect-level (NOAEL) was 5 mg/kg/day. Dosages of 20 and 50 mg/kg/day produced reductions in body weight and feed consumption. The developmental NOAEL was determined by the study authors to be 50 mg/kg/day. No developmental toxicity was produced at dosages as high as 50 mg/kg/day, the highest dose tested.

These two studies were considered as key study and the most relevant for teratogenicity assessment. Two additional studies with OECD 414 similar method were available. In one study (Burnett, et al., 1985 Klimish 2), female rats were treated at 0.003, 0.1 and 0.3% of test item in diet during 14 weeks of administration before mating (no treatment during mating) and were treated from day 0 to day 20 of gestation. Females were observed for signs of toxicity, uteri was analysed. Fetuses were observed for abnormalities. 0.3% test substance fed animals consistently weighed less and gained less weight than the control group throughout gestation. However, no statistically significant differences were noted. The mean group consumption of females fed 0.3% test substance was significantly lower than that of control. Based on the above findings, administration of N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulphate through diet to mated female Sprague-Dawley rats at dose levels of 0.3, 0.1 and 0.03% demonstrated a NOAEL of 0.1% in diet (equivalent to 98.04 mg/kg bw/d for female rats) for maternal and developmental toxicity. However, no teratological effects were observed at any dose levels tested.

Another study was available (Burnett C, 1976, Klimisch 2) in which females rats were dermally treated on days 1, 4, 7, 10, 13, 16 and 19 of gestation with two formulations : Formulation 1 (P21) containing 1% of test substance was prepared. Prior to treatment, the formulation was mixed with equal volume of 6% hydrogen peroxide; Formulation 2 (P22) contained 0.5% of test substance. No signs of toxicity were seen throughout the study. However, this study cannot be used for assessment due to significant deviations (treatment frequency should be daily, the doses of test item were too low).

Justification for classification or non-classification

Toxicity to reproduction:

The multigeneration reproduction study was designed to evaluate the reproductive toxicity of two formulation hair dyes containing N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate in Charles River CD rats.  Dermal application of P21 formulation containing 1% N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate mixed  with equal volume of hydrogen peroxide and dermal application of P22 formulation containing 0.5% of test substance to Charles River CD rats over three generations did not induce any treatment related effects on reproductive parameters in any of the generations. Additionally, a OECD 408 study was performed in which rats were treated orally for 90 fays to the test substance. Reproductive apparatus analysis was perfomed. No effect was observed on sperm motility and vaginal cytology. The NOAEL was defined as 20 mg/kg bw/day.

Based on the results of the available studies, the registered substance N,N-Bis-(2-hydroxyethyl)-p-phenylenediamine sulfate was Not Classified for Toxicity to Reproduction.

Developmental toxicity/ Teratogenicity :

The maternal and developmental toxicity potential of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate was determined following the OECD Guideline 414 (Prenatal Developmental Toxicity Study).

Administration of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate by oral gavage to mated female Crl:CD (SD)IGS BR VAF/Plus rats during gestation Day 6-20 at dose levels of 0, 5, 20 and 50 mg /kg bw/day revealed a NOAEL of 5 mg/kg bw/day for maternal toxicity and 50 mg/kg bw/day for developmental toxicity.  The registered substance was not classified for Teratogenicity.