4-amino-m-cresol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

From May 10, 1984 to July 18, 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Animals were treated during organogenesis phase Day 5-15 of gestation instead of guideline recommendation of dosing through the entire period of gestation to the day before caesarean section. the way of dose selection is not according to the OECD guideline and a possible hazard is not adequately identified (dose selection based on the in use concentration of the dye)

Justification for type of information:

See attached document in Annexe 1 : justification for read across analogy approach

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See Remark in "Rationale for reliability includ defiencies"

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

51956-65-1

Cas Number:

51956-65-1

IUPAC Name:

51956-65-1

Constituent 2

Reference substance name:

1-Hydroxy-3-methyl-4-aminobenzol-sulfat

IUPAC Name:

1-Hydroxy-3-methyl-4-aminobenzol-sulfat

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: 1-Hydroxy-3-methyl-4-aminobenzol-sulfat
- TSIN: Not reported
- Molecular formula: Not reported
- Molecular weight: Not reported
- Substance type: Pure active substance
- Physical state: Solid powder
- Stability under test conditions: As salt unlimitedly stable
- Storage condition of test material: At room temperature
- Solubility: In water

Test animals

Species:

rat

Strain:

Wistar

Remarks:

BOR:WISW-SPF TNO

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Fa. Winkelmann, Versuchstierzucht, Borchen, Germany
- Age at study initiation: 14 weeks old
- Weight at study initiation: Males: 300 g; Females: 166-228 g
- Housing: Animals were housed in Makrolon cages (Type II). The bedding used was from pure, spruce-, fir- and pine-wood, dried, disdusted and sterilized at 180°C.
- Diet: Pelleted rodent diet (Ssniff-R Alleindiat fur Ratten), ad libitum
- Water: Aqua Fontana, ad libitum
- Acclimation period: 14 days
- The quality of the diet was assumed by regular analysis by the producer. Organic and inorganic feed contaminants were analyzed quarterly.
- Quality was controlled by physical, chemical and bacteriological analysis twice yearly by an official laboratory for water analysis.

ENVIRONMENTAL CONDITIONS
- Temperature: 22±2°C. Registered twice daily in the morning and late afternoon
- Humidity: 45-85%. Registered twice daily in the morning and late afternoon
- Air changes: 16 times/hour. The air quality was of SPF-grade.
- Photoperiod: 12 hours of light and 12 hours of darkness per day

STUDY INITIATION DATE: May 10, 1984
STUDY COMPLETION DATE: July 18, 1984

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Deionized

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 100/400/800 mg of test substance was solved in 100 mL of deionized water to get the test concentrations for Group 1, 2 and 3, respectively.
- Rate of preparation of dose formulation: Daily

VEHICLE
- Concentration in vehicle: 0.1, 0.4 and 0.8%
- Amount of vehicle: 10 mL/kg

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: Not reported
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: A positive proof for sperms in the vaginal smear was taken as Day 0 of gestation. The vaginal smears were made in the early morning hours.

Duration of treatment / exposure:

Gestation days (GD) 5-15

Frequency of treatment:

Once daily

Duration of test:

11 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

80 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 mated females/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The maximal appointed concentration of the dyestuff is 3%. This means a maximal external exposition of about 36 mg per man by conditions of hair coloring. Considering an assumed maximal resorption of 0.2%, 0.072 mg intake per kg of bodyweight can be calculated. Therefore the lowest dosage of 10 mg will have a sufficient high safe zone to the appointed concentration. The producing of teratogenic effects by toxic doses should be proved by the application of the higher dosages. However, as per scientific committee on consumer products (SCCP /0898/05), this way of dose selection is not according to the OECD guideline and a possible hazard is not adequately identified.
- Rationale for animal assignment: Animals were randomized by allocating 12 males and 24 females from the collective to each test group, using computerized randomization tables.
- Rationale for test system: The rat was chosen for this study because of good fertility, high reproduction rates, good response to teratogens and a great number of background information.
- Rationale for route of administration: The oral route of administration was chosen to attain higher resorption rates compared to dermal exposure.
- Dose volume: 10 mL/kg bw
- Assignment of animals: The animals were assigned into following control and treatment groups:
Vehicle control: Deionized water
Group I: 10 mg/kg bw/day
Group II: 40 mg/kg bw/day
Group III: 80 mg/kg bw/day

Examinations

Maternal examinations:

MORTALITY : Yes
- Time schedule: Daily

CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
- Observations: Sensoric and motoric behavior considering hair coat, urine and fecal excretion and conditions of body orifices and special reflex examinations (modified to Irwin).

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0 (prior to treatment initiation), 5, 10, 15 and 20 of the study.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Food consumption was measured for phases 0-5, 5-15, 15-20 as well as for 0-20.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: 20, females were sacrificed by carbon dioxide asphyxiation.
- Organs examined: A complete autopsy of the dams and a macroscopic evaluation of the organs were carried out.

OTHER: The fetuses were developed by caesarian section.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number and distribution of implantation sites: Yes
- Number of live and dead fetuses: Yes
- Other: The weight of fetuses, placentae and uteri without fetuses was determined.

Fetal examinations:

- External examinations: Yes, all per litter. All fetuses were examined grossly for externally visible deviations from normal conditions.
- Soft tissue examinations: Yes, 1/3 litter. About 1/3 of all fetuses were selected at random, fixed in Bouin solution for at least 14 days and then deep frozen. For the evaluation of organic defects the slightly thawn fetuses were dissected according to the "free hand razor blade technique" (Wilson, modified) into approx. 1 mm thick slices. The pelvic cavity was evaluated directly
- Skeletal examinations: Yes, 2/3 litter. All fetuses after skin removal and alcoholic fixation, the skeletons were stained in Alizarin. The evaluation of skeletal defects was done directly using a magnifier.
- Head examinations: No

Statistics:

- One factorial analyses of variance were calculated for growth parameters and fetal parameters (number of fetuses, implantations, corpora lutea, weights of fetuses and placentae). The comparison of groups mean values was carried out according to the method of "TUKEY".
- Indices, if necessary, were compared with the U-test of Mann-Whitney.
- Significance levels are noted as follows:
* p < 0.05: Slightly significant
** p < 0.01: Significant
*** p < 0.001: Highly significant

Indices:

a) Gestation index = (Litters/pregnant dams) × 100
b) Still birth index = (Resorptions/implantations) × 100
c) Variation index = (Variations/total number of fetuses) × 100
d) Malformation index = (Malformations/total number of fetuses) × 100
e) Runts index = (Runts/total number of fetuses) × 100
g) Post implantation loss index = ((implantations-fetuses alive)/implantations) × 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Gross pathological findings:

no effects observed

Maternal developmental toxicity

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

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY:
- No mortality was observed in the study.

MATERNAL BODY WEIGHTS AND BODY WEIGHT CHANGES:
- Bodyweight gains did not show significant intergroup differences in any of the evaluated phases.

MATERNAL FOOD CONSUMPTION:
- Food consumption revealed no significant intergroup differences.

CLINICAL OBSERVATIONS:
- No treatment-related effects in dams were noted with regard to clinical observations.
- The haircoat of all females in control and dose groups appeared smooth and brightly. Animals showed normal excretion of urine and feces and a healthy general status.
- Examinations of ears, hearing and reflexes revealed no deviations from normal findings.

MACROSCOPIC FINDINGS (Autopsy):
- No gross signs of organ alterations were found that could be attributed to treatment.

LAPAROTOMY: The results were as follows:
- Number of Fetuses: The test groups showed no significant differences concerning the mean number of fetuses per dam.
- Left - Right Intrauterine Distribution: Mean number of fetuses per dam in the left or right uteri horn was not dose related different among groups.
- Sex Ratio: There were no significant differences in the sex ratio.
- Birth position: No significant differences were determined among groups, concerning the number of fetuses in anterior or posterior position within uteri.
- Runts: There were only one runt in control group and one in Group III.
- Dead Fetuses and Life Birth Index: One fetus from Group III was found dead. The life birth index in this group was therefore slightly reduced, but this was within the normal variation range in rats.
- The life birth index for Group I, II and III was 100, 100 and 99.4%, respectively. The life group index in control group was 100%.
- Number of Resorptions: Mean number of resorptions showed no significant differences in the test groups.
- Indices of Resorptions: Distinct differences between groups were not found.
- Implantations: Mean number of total implantations showed no significant differences between test groups. The number of implantations in the left uterine horn was slightly significant reduced within Group I and II females, which is an incidental finding by all means.
- Post-Implantation loss Index: No significant differences among test groups were noticed.
- Corpora lutea: Dose related intergroup differences were not observed (neither in the total number nor in the number of the left or right ovary).
- Placenta Weight: There were no significant intergroup differences.
- Weights of Gravid Uteri: Significant intergroup differences were not determined.
- Weights of Uteri: Significant intergroup differences were not determined.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

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

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

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:
WEIGHT OF FETUSES:
- Mean weights of fetuses showed no essential differences between control and dose groups.

EXTERNAL EXAMINATIONS:
- The external examination did not reveal malformations in the evaluated fetuses.

FETAL SOFT TISSUE ALTERATIONS:
- In Group II, one fetus with hydrocephaly internal malformation was observed.
- In Group III, one fetus with an increased renal pelvic cavitation in the right kidney (minor visceral anomalies) and 2 fetuses with an increased renal pelvic cavitation in the left kidneys (minor visceral anomalies) were observed.

FETAL SKELETAL ALTERATIONS:
- Examinations of the stained fetal skeletons did not reveal signs of retarded ossifications of the skull bones and the thoracic vertebral centres. Sizes of fontanelles were mainly small in all groups and rib number was 13 in both sides of all fetuses.
Malformation Index:
- Due to the fetus with a hydrocephaly in Group II the index in this group was slightly increased (0.56%).

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Overall effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Administration of 1-Hydroxy-3-methyl-4-aminobenzol-sulfat to female BOR:WISW-SPF TNO rats during Day 5-15 of gestation, at dose levels of 0, 10, 40 and 80 mg/kg bw/day by oral gavage revealed a NOAEL of 80 mg/kg bw/day for maternal and developmental toxicity.

Executive summary:

The teratology study of 1-Hydroxy-3-methyl-4-aminobenzol-sulfat was conducted following methods comparable to the OECD Guideline 414 (Prenatal Developmental Toxicity Study).

The study was designed to assess the effects of the test substance on pregnant female and on embryonic and fetal development when administered by oral gavage once daily at dose levels of 0, 10, 40 and 80 mg/kg bw/day (Dose volume: 10 mL/kg bw) from Day 5 through to Day 15 of gestation.

Female BOR:WISW-SPF TNO 14 week old rats (Source: Fa. Winkelmann, Versuchstierzucht, Borchen, Germany) weighing 166-228 g were housed in Makrolon cages. The animals were maintained under standard laboratory conditions (temperature: 22±2 °C, humidity: 45 -85%, air changes: 16 times/hour and 12 hours of light and 12 hours of darkness per day). Animals were fed on Pelleted rodent diet, ad libitum. The animals were acclimatized for 14 days.

Prior to acclimation, females were paired with males (2:1) until mating was confirmed. Presence of sperm in vaginal smear was considered to be proof of pregnancy and the day of observation was referred to as Day 0 of presumed gestation (GD 0).

Each treatment and control group contained 24 mated females.

All animals were observed daily for mortality and clinical observations. Body weights were recorded on Days 0 (prior to treatment initiation), 5, 10, 15 and 20 of the study. Food consumption was measured for phases 0-5 days, 5-15 days, 15-20 days as well as for 0-20 days.

On gestation Day 20, the dams were sacrificed and examined for visceral gross pathology, cesarean-sectioned, and evidence of maternal and developmental toxicity.

The ovaries and uterine content (gravid uterus weight, number of corpora lutea, number of implantations, numbers of early and late resorptions, number and distribution of implantation sites) was examined after sacrifice.

Numbers of live and dead fetuses were determined. Each fetus was weighed, sexed and examined externally. Fetuses were examined for visceral and skeletal alterations.

No mortality was observed in the study. No treatment-related effects in dams were noted with regard to clinical observations and postmortem findings. The body weight and the food consumption were not affected by the treatment. Gross necropsy revealed no treatment related effects.

There were no treatment related effects with regard to uterus and placenta weights, the number of corpora lutea, and implantations.

There were no treatment related effects with regard to reproduction e.g. litter size, foetal mortality, foetal body weight, birth position and sex ratio. The skeletal and visceral examination of the fetuses revealed no treatment related findings. Neither a statistically significant difference as compared to the concurrent control nor a dose-dependent increase in any malformation or variation was noted.

In conclusion, administration of 1-Hydroxy-3-methyl-4-aminobenzol-sulfat to female BOR:WISW-SPF TNO rats during Day 5-15 of presumed gestation, at dose levels of 0, 10, 40 and 80 mg/kg bw/day by oral gavage revealed a NOAEL of 80 mg/kg bw/day for maternal and developmental toxicity.