2-(2H-benzotriazol-2-yl)-p-cresol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The substance is considered to cause no adverse effects on fertility based on the absence of histopathology findings in organs related to male and female reproduction, absence of distribution into organs other than liver and kidney upon subchronic exposure (Knight 2007) as well as on the absence of findings in a screening study (OECD 422) (MHLW 2007).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Japan, Inc. (Atsugi Breeding Center)
- Age at study initiation: 10 -11 weeks
- Weight at study initiation: 370 to 422 g in males, 225 to 263 g in females
- Fasting period before study: none
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): 15 to 17
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dosing samples were prepared once a week based on the results of the 8-day stability study conducted at the Japan Bioassay Research Center and stored in a dark place at room temperature and were used within 7 days after preparation.


VEHICLE
- Justification for use and choice of vehicle (if other than water): The substance is not soluble in water, but suspendable in olive oil
- Concentration in vehicle: adjusted to volume of 5 ml/kg bw
- Amount of vehicle (if gavage): 5 ml/kg bw

Details on mating procedure:

The males and females of the same group were mated by cohabitation in 1:1 sex ratio every day for 14 days at the longest starting from Day 15 of treatment. Copulation was confirmed by the presence of vaginal plug in the vagina or presence of sperms in the vaginal smear and the day when these were found was defined as Day 0 of gestation. The females with establishment of copulation were promptly separated from males and housed individually.
The time to copulation (number of days taken from the start of mating to establishment of copulation) and the copulation rate [(number of pairs that established copulation / number of pairs mated) x 100] were calculated based on the results of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance was checked by comparing the chromatograms determined using high-performance liquid chromatography (Hewlett Packard 1090) before and after use. The concentration and uniformity of the test substance in the dosing samples were checked by the measurement using high-performance liquid chromatography (Hewlett Packard 1090) at the time of the initial preparation.

Duration of treatment / exposure:

Males, 42 days
Females, from 14 days before mating to day 4 of lactation
Females (satellite), 42 days

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Males, 12 (5 for recovery)
Females, 12; satellite females, 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose selection rationale: The doses in this study were determined based on the results of the preliminary study. The dose levels in the preliminary study were determined by setting 1000 mg/kg as the highest dose level as specified in the OECD Guideline for Testing of Chemicals, followed by 300 mg/kg, 100 mg/kg, and 30 mg/kg.

Five each of male and female Crl:CD(SD) rats aged 10 weeks per group received 2-(2’-hydroxy-5’-methylphenyl) benzotriazole orally for 14 consecutive days and were autopsied on the day after completion of administration. As a result, the test substance had no effect on the body weight or general health condition of the males and females in all treatment groups. Both the males and females given 100 mg/kg and higher showed an increase in liver weight and the females also showed an increase in total cholesterol. Increased phospholipid was also noted in the females given 300 mg/kg and higher. Based on these results, 300 mg/kg was selected as the high dose level in this study followed by 100 mg/kg as the intermediate dose level and 30 mg/kg as the low dose level.

Positive control:

not required

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week
In the observation of females in Week 6 of treatment, however, only the satellite animals were observed to reduce the burden on the animals due to parturition and nursing.
The animals were observed for contact reaction, vocality and easiness of taking out when they were taken out from cages, easiness for handling, body temperature, fur condition, skin color, eye condition and salivation after taking out from cages, posture, activity, alert/searching behavior, gait condition, stereotypical behavior, respiration, and tremor/spasm as well as numbers of events of urination and defecation, grooming and face washing per minute on the work table.

BODY WEIGHT: Yes
- Time schedule for examinations: The males were weighed on Days 1, 8, 15, 22, 29, 36 and 42 of treatment, and the recovery animals were weighed on Days 1, 8 and 14 of recovery.
All the females were weighed on Days 1, 8 and 15 of treatment before mating. After the start of mating, they were weighed on Days 0, 7, 14 and 20 of gestation, on the parturition day and Day 4 after parturition (day 0 and 4 of nursing). The satellite females were weighed on Days 1, 8, 15, 22, 29 and 36 of treatment and Days 1, 8 and 14 of recovery.
On the autopsy day, the body weight was measured in all animals after fasting.

Food consumption: Yes
For the males, food consumption was measured between Days 1 and 8, 8 and 15, 29 and 36, and 36 and 42 in the treatment period and between Days 1 and 8, and 8 and 14 in the recovery period.
For the females, food consumption was measured for all animals before mating between Days 1 and 8, and 8 and 15. After the start of mating, food consumption was measured between Days 0 and 7, 7 and 14, 14 and 20 of gestation and between Days 0 and 4 of nursing. For the satellite females, food consumption was measured between Days 1 and 8, 8 and 15, 29 and 36, and 36 and 43 of treatment and between Days 1 and 8, and 8 and 14 of recovery.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at autopsy
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: all
- Parameters checked: Red blood cell count, platelet count, reticulocyte ratio, white blood cell count, mean corpuscular volume (the light scattering method, hereinbefore), hemoglobin concentration (cyanmethemoglobin method), hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration (by calculation, hereinbefore) were determined by the general hematology system (ADVIA 120: Bayer), differential leukocyte classification (by Wright’s stain) using the automatic blood cell analyzer (MICROXHEG-120NA: Omron), and prothrombin time (Quick one-step method) and activated partial thromboplastin time (ellagic acid activation method) using the full automatic blood coagulation tester (Sysmex CA-5000: Sysmex).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at autopsy
- How many animals: all
- Parameters checked: for total protein (biuret method), albumin (BCG method), A/G ratio (calculation), total bilirubin (azobilirubin method), glucose (GlcK･G-6-PDH method), total cholesterol (CE･COD･POD method), triglyceride (MGLP･GK･GPO･POD method), phospholipid (PLD･ChOD･POD method), AST, ALT, γ-GTP, CK (JSCC method, hereinbefore), LDH (SFBC method), ALP (GSCC method), urea nitrogen (urease GLDH method), creatinine (Jaffé method), sodium, potassium, chlorine (ion-selective electrode method, hereinbefore), calcium (OCPC method) and inorganic phosphorus (PNP･XOD･POD method) using an automatic analyzer (Hitachi 7080: Hitachi, Ltd.).

URINALYSIS: Yes
Fresh urine was collected before administration from all males and satellite females in Week 6 and pH, protein, glucose, ketone bodies, bilirubin, occult blood and urobilinogen were tested using a urine test paper (Multistix, Bayer).
Urinalysis was also performed for all male and female recovery animals in Week 2 of recovery as changes suspected of the effect of the test substance administration were found in the males given 100 mg/kg and higher.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: All the males were tested in Week 6 of treatment. As for the females, 5 animals that underwent parturition on the same day or closer days were selected in each group and the tests were performed on the day before autopsy in the combined repeated-dose oral toxicity and reproductive and developmental toxicity study (day 4 after parturition).
- Battery of functions tested: sensory activity, grip strength and motor activity
In the reactivity test, vision, hearing, pain sensation, pupil reflex and aerial righting reflex were examined.
Grip strength was measured using a grip strength meter (MK-380CM, Muromachi Kikai Co., Ltd.). Both the forelimbs and hindlimbs were measured twice and the means were defined as the grip strengths of the individuals.
Locomotor activity was measured for 60 minutes per animal using a locomotor measuring instrument (SCANET MV-10, Melquest).

Oestrous cyclicity (parental animals):

Vaginal smears were collected from all the females (excluding satellite females) every morning from the start of treatment to the first day of mating (day 15 of treatment), stained with Giemsa stain, and observed for sex cycle under a light microscope.

Sperm parameters (parental animals):

none

Litter observations:

The body weight of pups was measured per dam (litter) on Days 0 and 4 of nursing by the units of males and females and the mean per pup was calculated.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

The ovaries were excised from copulated females at the time of autopsy and the number of corpus luteum verum was counted under a stereomicroscope. In addition, the uterus was excised, stained with 10% ammonium sulfate solution, and implantation sites were counted on the following day.
The implantation rate [(number of implantation sites / number of corpus luteum verum) x 100] was calculated based on these results.


GROSS PATHOLOGY: Yes
Autopsy was performed for the males excluding recovery animals on the day after Day 42 of treatment and for the recovery animals on the day after Day 14 of recovery. The females that underwent parturition were autopsied on Day 5 after parturition and those that did not give parturition were autopsied on the day corresponding to Day 26 of gestation to check presence or absence of conception. The satellite females were autopsied on the day after Day 14 of recovery.

HISTOPATHOLOGY: Yes
The testes, epididymides, seminal vesicles/coagulating gland, prostate (ventral lobe) and ovaries of all animals and trachea, lungs, bone marrow (femur), lymph nodes (axillary, peritoneal, etc.), thymus, spleen, heart, stomach, small intestines (including duodenum), large intestines, liver, kidneys, bladder, pituitary body, thyroid gland, parathyroid gland, adrenal glands, uterus (only in the satellite females), vagina, mammary glands, brain, spinal cord, peripheral nerve (sciatic nerve), muscle, and bone (femur) of 5 animals in each group (in the ascending order of animal numbers, excluding infertile females) and recovery animals were excised, embedded in paraffin, sectioned, and stained in hematoxylin and eosin, and observed under a light microscope.

Postmortem examinations (offspring):

GROSS NECROPSY
All the pups that survived on Day 4 of nursing were laparotomied under ether anesthesia, exsanguinated by cutting the abdominal aorta, and autopsied. The pups that died in the course were also autopsied.

Statistics:

The chi-square test was performed for the copulation rate, conception rate, birth rate, urinalysis and histopathology results between the control group and each of the treated groups.
For the numeric data obtained in other tests, equal variation was examined preliminary by the Bartlett method using the control group as the basis. The one-way analysis of variance was performed if equal variation was shown and the Dunnett’s multiple comparison was performed for testing the means when a significant difference was detected between the groups. If the variation was not equal, the measured values were ranked across the groups and the Kruskal-Wallis rank test was performed followed by the Dunnett-type multiple comparison if a significant difference was detected between the groups. In the statistical analysis of the numeric data in the recovery animals, the F-test was performed first followed by the Student’s t-test if there was no difference in distribution and by the Aspin-Welch’s t-test if the distribution showed a difference.
The significance level was set at 5% and 1% on both sides and 5% significance level was set as the judgment criteria for the effect of the test substance

Reproductive indices:

Copulation index = (No. of copulated pairs / No. of mated pairs) × 100.
Fertility index = (No. of pregnant females / No. of copulated pairs) × 100.
Gestation index = (No. of pregnant females with live pups / No. of pregnant females) × 100.
Implantation index = (No. of implantations / No. of corpora lutea) × 100.
Delivery index = (No. of pups born / No. of implantations) × 100.

Offspring viability indices:

Viability index = (No. of live pups on day 4 / No. of live pups on day 0) × 100.
Birth index = (No. of live pups on day 0 / No. of implantations) × 100.
Live birth index = (No. of live pups on day 0 / No. of pups born) × 100.
Sex ratio on day 0 = No. of male live pups on day 0 / No. of live pups on day 0.
Sex ratio on day 4 = No. of male live pups on day 4 / No. of live pups on day 4.

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

A NOAEL of 30 mg/kg bw was defined based on the increase in relative liver weight for males. In the histological examination, liver and kidney lesions occurred in the dosed group of both sexes. The hepatic lesions were characterized by increased incidences of hypertrophy of the centrilobular hepatocytes in the males given 300 mg/kg and in the females given 100 mg/kg and above. Theses changes were not observed at the end of the recovery period. The kidney lesions exhibited a gender difference, as characterized by increased incidences of an eosinophilic body of proximal tubules in the males given 300 mg/kg, and hydropic change and regeneration of proximal tubules in the females given 100 mg/kg and above. The eosinophilic body of proximal tubules remained affected in the recovery group of males given 300 mg/kg, whereas female kidney lesions were not observed in the satellite females given 300 mg/kg.



No adverse effects on reproductive performance was observed for parental animals at the highest tested dose of 300 mg/kg bw.

Dose descriptor:

NOEL

Effect level:

>= 30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects on liver at clincial chemistry observed in dose-range finding study at 1000 mg/kg bw

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

kidney

liver

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

No effect of the compound was observed on the reproductive performances, such as the estrous cycle, copulation, fertility, delivery and lactation. No effect of the compound was observed on the implantation, the number of pups, sex ratio of pups, viability of the pups during 4 days of lactation and weights of the pups. No external abnormalities or macroscopic findings were detected in any pup.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: as far as F1 can be assessed in a screening study.

Critical effects observed:

no

Reproductive effects observed:

no

Table 1: Estrous cycle and reproductive performance

Dose (mg/kg)	0	30	100	300
Estrous cycle
No. of animals examined	12	12	12	12
Type of cycle
4-day	10	9	9	12
5-day	2	2	1	0
Others Length (day)a)	0 4.2±0.4	1 4.2±0.4	2 4.2±0.4	0 4.0±0.0
Reproductive performance
No. of mated pairs	12	12	12	12
No. of copulated pairs	12	12	12	12
Copulation index (%)	100	100	100	100
No. of pregnant females	12	12	12	12
Fertility index (%) Pairing days until copulationa)	100 2.3±1.1	100 4.0±3.1	100 2.3±1.2	100 2.4±1.2
Parturition and lactation
Non-remarkable	12	12	12	11
Abnormal lactation	0	0	0	1

a) Data represent mean ± S.D.

Copulation index = (No. of copulated pairs / No. of mated pairs) × 100.

Fertility index = (No. of pregnant females / No. of copulated pairs) × 100.

Table 2 Summary of development of pups from dams treated orally with 2-(2'-hydroxy-5'-methylphenyl)benzotriazole in the combined repeated dose and reproductive/developmental toxicity screening test

Dose (mg/kg)	0		30		100		300
No. of pregnant females	12		12		12		12
No. of pregnant females with live pups	12		12		12		12
Gestation index (%)	100		100		100		100
Gestation length (days)	21.9±0.5	(12)	21.9±0.3	(12)	22.1±0.3	(12)	21.9±0.3	(12)
No. of corpora lutea	16.0±1.3	(12)	16.2±1.2	(12)	16.0±2.0	(12)	16.3±1.7	(12)
No. of implantations	15.3±1.5	(12)	14.7±1.8	(12)	15.0±1.3	(12)	15.4±1.4	(12)
Implantation index (%)	95.2±2.9	(12)	90.8±9.5	(12)	94.5±9.3	(12)	95.1±5.5	(12)
Day 0 of lactation
No. of pups born	14.4±1.4	(12)	13.6±2.0	(12)	13.5±2.8	(12)	14.0±1.4	(12)
Delivery index (%)	94.7±6.5	(12)	92.8±9.5	(12)	90.0±17.0	(12)	91.0±6.8	(12)
No. of live pups	14.3±1.5	(12)	13.5±2.0	(12)	13.4±3.1	(12)	13.8±1.5	(12)
Birth index (%)	93.6±8.0	(12)	92.2±9.3	(12)	89.4±18.8	(12)	89.5±9.0	(12)
Live birth index (%)	98.7±3.0	(12)	99.4±2.0	(12)	98.6±4.8	(12)	98.2±3.3	(12)
Pups weight (g)
Male	6.6±0.4	(12)	6.5±0.5	(12)	6.8±0.5	(12)	6.8±0.5	(12)
Female	6.3±0.5	(12)	6.1±0.4	(12)	6.5±0.4	(12)	6.4±0.4	(12)
Sex ratio on day 0	0.56±0.12	(12)	0.48±0.10	(12)	0.50±0.16	(12)	0.50±0.10	(12)
Day 4 of lactation
No. of live pups	14.2±1.6	(12)	13.3±2.2	(12)	13.2±2.9	(12)	12.6±3.9	(12)
Viability index (%)	99.4±2.2	(12)	98.0±4.9	(12)	98.4±3.9	(12)	91.7±26.7	(12)
Pups weight (g) Male	10.3±1.0	(12)	10.5±1.2	(12)	10.8±1.6	(12)	10.5±0.8	(11)
Female	9.9±1.1	(12)	9.9±1.2	(12)	10.4±1.5	(12)	9.5±1.8	(12)
Sex ratio on day 4	0.56±0.12	(12)	0.48±0.11	(12)	0.50±0.16	(12)	0.46±0.18	(12)

Data represent mean ± S.D.

Parentheses represent the number of litters examined.

Gestation index = (No. of pregnant females with live pups / No. of pregnant females) × 100.

Implantation index = (No. of implantations / No. of corpora lutea) × 100.

Delivery index = (No. of pups born / No. of implantations) × 100.

Birth index = (No. of live pups on day 0 / No. of implantations) × 100.

Live birth index = (No. of live pups on day 0 / No. of pups born) × 100.

Sex ratio on day 0 = No. of male live pups on day 0 / No. of live pups on day 0.

Viability index = (No. of live pups on day 4 / No. of live pups on day 0) × 100.

Sex ratio on day 4 = No. of male live pups on day 4 / No. of live pups on day 4.

Conclusions:

No indication of reproductive toxicity by the oral route was identified in this screening study.

Executive summary:

No effect of the compound was observed on the reproductive performances, such as the estrous cycle, copulation, fertility, delivery and lactation. No effect of the compound was observed on the implantation, the number of pups, sex ratio of pups, viability of the pups during 4 days of lactation and weights of the pups. No external abnormalities or macroscopic findings were detected in any pup.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A screening study for reproductive toxicity (OECD 422) was performed in 2006, and a summary report was published in 2007 in Japanese the sponsor MHLW Japan (MHLW 2007). This summary report was translated into English. Although it is not a full study report, it contains sufficient information for evaluation and interpretation of the study. The study is indicated to be GLP and OECD guideline compliant. It was performed with gavage dosing of 30, 100 and 300 mg/kg bw using olive oil as vehicle and doses were based on systemic toxicity on parental animals. A NOAEL of 30 mg/kg bw was shown for systemic toxicity of parental animals. No effect of the compound was observed on the reproductive performances, such as the estrous cycle, copulation, fertility, delivery and lactation. No effect of the compound was observed on the implantation, the number of pups, sex ratio of pups, viability of the pups during 4 days of lactation and weights of the pups. No external abnormalities or macroscopic findings were detected in any pup.

The substance did not cause adverse effects on reproductive organs upon subchronic and chronic exposure feeding studies. For details it is referred to the chapter on repeated dose toxicity.

Taken together, the substance is not considered to cause adverse effects on fertility.

Short description of key information:
The substance is considered to cause no adverse effects on fertility based on the absence of histopathology findings in organs related to male and female reproduction, absence of distribution into organs other than liver and kidney upon subchronic exposure (Knight 2007) as well as on the absence of findings in a screening study (OECD 422) (MHLW 2007).

Effects on developmental toxicity

Description of key information

No developmental toxicity and teratogenicity was observed in two studies with rats and mice (Fritz 1975b and c). The design of both studies is sufficiently similar to OECD testing guideline 414 and the reporting details are adequate for study assessment. Both studies were performed prior to the introduction of GLP. 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not indicated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study design appears to follow OECD Guideline 414 (2001). Study pre-dates GLP requirements. No analytical verification of dose concentrations (considered acceptable, as substance was found stable in other studies).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

treatment only from day 6 - day 15

GLP compliance:

no

Remarks:

Study pre-dates GLP.

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): TK 10047 (Tinuvin P)

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: closed SPF breeding colony (facility name not reported).
- Weight at study initiation: about 240 g (females).
- Housing: successfully mated females kept in groups of 5 in Macrolon cages.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 to 22.5 degrees Celcius.
- Humidity (%): 51 to 61%.
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

VEHICLE
- Amount of vehicle (if gavage): the amount of fluid administered was 1 mL/100 g of body weight.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused.
- If cohoused:
- M/F ratio per cage: 1:3
- Length of cohabitation: overnight.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.

Duration of treatment / exposure:

From Day 6 until Day 15 of pregnancy, inclusive.

Frequency of treatment:

Once/day.

Duration of test:

Females were dosed from Day 6 until Day 15 of pregnancy (inclusive) and dams were autopsied and foetuses removed by Caesarean section on Day 21.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 females/dose group and 30 females in the control group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: None reported.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily.

BODY WEIGHT: Yes
- Time schedule for examinations: daily.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: As per study report, "The examinations were carried out in accordance with W.H.O. recommendations (Wld. Hlth. Org. Techn. Rep. Ser. 364, 1967)." Organs examined included the ovaries and uterus (mucosa and contents, including amniotic fluid and placentae).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 of the foetuses were fixed in a mixture of alcohol and formol to which acetic acid was added.
- Skeletal examinations: Yes: 2/3 of the foetuses following clearing in potassium hydroxide and staining with Alizarine Red S.

Statistics:

Not reported.

Indices:

Not reported.

Historical control data:

yes

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Slight increase in feed consumption was noted towards the end of treatment.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No adverse effects on embryonic and foetal development were noted, when data on the rates of implantations and embryolethality (resorptions) as well as foetal average weight were compared with the corresponding data of the CMC-control. By applying the slicing technique few minor anomalies were detected. The incidence of anasarca (slight oedema-like changes of siabcutis) was 5% at the highest dose group compared to 1% in the cumulative control and is still within the 99 % confidence limits of the CMC-control.
Skeletal assessment did not reveal any clear-cut deviation from the CMC-control except for a slight decrease in the number of not yet ossified phalangeal nuclei of the hind-limb as well as in the number of still incompletely ossified sternebrae at the mid dose only.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

The substance causes no teratogenicity, embryotoxicity, developmental toxicity and maternal toxicity in rats.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

no

Remarks:

Study pre-dates GLP.

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): TK 10047 (Tinuvin P)
- Batch: EN 2790

Species:

mouse

Strain:

NMRI

Details on test animals or test system and environmental conditions:

The albino mice were NMRI-derived and obtained from a closed SPF breeding colony. The initial body-weight of the females was about 30 g. They were mated overnight with males of proven fertility at a ration of 1 male to 4 females. The day on which a vaginal plug was observed was designated as "Day 0" of pregnancy. Throughout the experiment the successfully mated' females were kept in groups of 5 in Macrolon cages in an air-conditioned room at a temperature of 22° C + 0.5° C and a humidity of 56% + 5%. The room was illuminated for 12 hours daily. The standard diet fed was Nafag No. 890; tap-water was available at all times.

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

other: 2% Carboxymethylcellulose in water

Details on exposure:

The amount of fluid administered was 0.1 ml/10g of body-weight.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:4
- Length of cohabitation: overnight.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.

Duration of treatment / exposure:

From Day 6 until Day 15 of pregnancy, inclusive.

Frequency of treatment:

Once/day.

Duration of test:

Females were dosed from Day 6 until Day 15 of pregnancy (inclusive) and autopsy and removal of the foetuses by Caesarean section occurred on Day 18.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

30 females/dose group and 60 females in the vehicle group.

Control animals:

yes, concurrent vehicle

Details on study design:

During the period of treatment, general condition, weight-gain and symptomatology were checked daily. Feed consumption was noted.

Maternal examinations:

Dams were autopsied and foetuses removed by Caesarean section on Day 18 of pregnancy.
The examinations were carried out in accordance with W.H.O. recommendations (Wld. Hlth. Org. Techn. Rep. Ser. 563, 1975).

Ovaries and uterine content:

yes

Fetal examinations:

Following assessment of the dam's organs, especially of the ovaries and uterus (mucosa and contents, including amniotic fluid and placentae), the foetuses were subjected to careful external inspection and the condition of their body orifices was checked. They were then weighed individually and submitted to the following procedures:
1. Examination of the viscera according to the slicing
technique of WILSON*): 1/3 of the foetuses were fixed in a mixture of ethyl alcohol and formol to which acetic acid was added.
2. Skeletal assessment in 2/3 of the foetuses following clearing in potassium hydroxide and staining with Alizarine Red S.

Statistics:

performed, but method not given

Historical control data:

yes

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Markedly increased feed intake at the high-dose level as well as slightly decreased feed intake at the intermediate dose level indicate some compound-related reaction to treatment in the dams. The dams of the low-dose group remained entirely unaffected by the administration of TK 10047.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: absence of adverse effects

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Following the low-dose level phalangeal nuclei of the fore-limb and sternebrae showed complete ossification at higher incidences than the CMC-control. Since this finding is not correlated with any changes of foetal development and could not be observed atthe intermediate, and high dose level the significance of this finding is not apparent.

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

The substance is does not cause maternal toxicity, teratogenicity, developmental toxicity or embryotoxicity in mice.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

For developmental toxicity and teratogenicity, two valid rodent studies are available, and both of them can serve as key studies. The design of both studies is sufficiently similar to OECD testing guideline 414 and the reporting details are adequate for study assessment. Both studies were performed prior to the introduction of GLP.

In the teratology study in rats (Fritz 1975b) a total of 105 Sprague-Dawley pregnant females (25 per dose group, except control with 30) were dosed by gavage with 0, 150, 500 and 1000 mg/kg bw from day 6 through 15 of pregnancy.

In the mouse teratology study (Fritz 1975c) a total of 150 NMRI derived (SPF) females (30 per dose group, except control with 60) were dosed by gavage with 0, 150, 500 and 1000 mg/kg bw from day 6 through 15 of pregnancy.

There were no treatment related reactions noted, with the exception of the mouse study where increased feed intake at the high dose level as well as a slightly decreased feed intake at the intermediate dose level was noted. Embryonic development was not impaired and no teratogenic effects were noted.

In addition, no indication of developmental toxicity was observed in the screening study (OECD 422) performed with gavage application using olive oil as vehicle (MHLW 2007). For this study, 300 mg/kg bw was used as the highest dosed.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for reproductive toxicity under Regulation (EC) No. 1272/2008.