2-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. Due to read-across from CAS 2440-22-4 the reliability was set to 2.

Data source

Reference

Reference 1

Reference Type:

other: new translation of publication

Title:

Unnamed

Year:

2012

Report date:

2012

Reference 2

Reference Type:

publication

Title:

English Abstract of OECD 422 study in Japanse language with CAS 2440-22-4

Author:

METI

Year:

2006

Bibliographic source:

http://www.meti.go.jp/

Materials and methods

Test guideline

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:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

219-470-5

EC Name:

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

Cas Number:

2440-22-4

Molecular formula:

C13H11N3O

IUPAC Name:

2-(2H-benzotriazol-2-yl)-4-methylphenol

Test material form:

solid

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-(2’-Hydroxy-5’-methylphenyl) benzotriazole
- Analytical purity: 99.9%
- Lot/batch No.: DWG7396
- Storage condition of test material: room temperature in the dark

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center of Charles River Laboratories Japan, Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: range - males: 370 - 422 g, females: 225 - 263 g
- Housing: individual in stainless-steel wire mesh cages, dams after day 18 of gestation were housed in special cages with bedding
- Diet: solid feed (CRF-1, 30 kg sterilized by gamma irradiation, Oriental Yeast Co., Ltd.), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulation (suspension) was prepared by mixing the olive oil with the test substance such that single dose liquid volume for each dose amounts to 5 mL/kg body weight. The dose formulation was prepared at one frequency per week based on the result of the stability test conducted by Japan Bioassay Research Center (JBRC) and stored at room temperature in a dark place to be used within 7 days after preparation.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. : 5915

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: sperm found in vaginal plug or vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged: individual housing

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and uniformity of the test substance in the dose formulation were measured at the time of its first preparation by the use of a high-performance liquid chromatography (HPLC: Hewlett Packard 1090) to verify them.

Duration of treatment / exposure:

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

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30, 100 and 300 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Males: 12 (5 for recovery)
Females: 12 + 5 satellite females (not mated)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected based on a preliminary study (oral gavage, doses: 30, 100, 300 and 1000 mg/kg bw) for 14 days. As a result, no effects of the administration on the body weights and general conditions of all the male and female groups administered were found. Increased liver weight was found in both males and females of 100 mg/kg and higher dose groups, and increased total cholesterol was also found in females. In addition, increased phospholipid was found in females of 300 mg/kg and higher dose groups. According to these results, 300 mg/kg was set as higher dose, and 100 and 30 mg/kg were set as medium and lower doses, respectively.

- Post-exposure recovery period in satellite groups: 14 days

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS + DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (before and after administration), once daily during recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: male: days 1, 8, 15, 22, 29, 36 and 42 and recovery animals on day 1, 8 and 14 of recovery period; female: before mating on days 1, 8 and 15; after start of mating on days 0, 7, 14 and 20 of gestation; on days 0 and 4 of lactation; satelite females were weighed on days 1, 8, 15, 22, 29, 36 and on days 1, 8 and 14 of recovery period

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

Oestrous cyclicity (parental animals):

Vaginal smears of all the females (except for the satellite females) were sampled every morning from the start day of administration to the start day of mating (day 15 of administration) and subjected to Giemsa stain to observe their estrous cycles by optical microscope.

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations:
testis weight, epididymis weight

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups and appearance were checked at birth, general condition and number of offspring every day until day 4 of lactation

GROSS EXAMINATION OF DEAD PUPS:
yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [after 42 days of treatment]
- Maternal animals: All surviving animals [day 4 of lactation]

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
Ovaries of the copulated females were removed at their necropsy to count the number of corpora lutea using stereo-microscope. In addition, uterus of the copulated females was removed at their necropsy and stained with a 10% solution of ammonium sulfide to count the number of
implantation sites on the following day.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: laparotomy under ether-anesthesia and killed by exsanguination followed by necropsy

Statistics:

Chi-square test was conducted for histopathological examinations. Data obtained for all other examinations was first tested for equality of variances using Bartlett test. One-way analysis of variance was used for equal variances followed by Dunetts multiple comparison when significant diferences were found among the groups. For unequal variances Kruskal-Wallis ranking test followed by Dunetts multiple comparison was applied.
Data from the recovery animals was first subjected to F-test and statistical processing was conducted using students t-test (equal variances) or Aspin-Welch's t-test (unequal variances). Significance was tested for confidance intervals of 5% and 1%.

Reproductive indices:

Copulation index (%): (Number of copulated animals / number of mating animals) x 100
Fertility index: (Number of pregnant females / (Number of copulated animals) x 100
Delivery index: (Number of pregnant females which delivered live offspring/Number of pregnant females) x 100
Implantation index (%): (Number of implantation sites / Number of corpora lutea) x 100

Offspring viability indices:

Delivery index: (Number of pups delivered / Number of implantation sites) x 100
Live birth index: (Number of live pups on day 0 of lactation / Number of implantation sites) x 100
Viability index (%) on day 4 of lactation: (Number of live pups on day 4 of lactation / Number of pups on day 0 of lactation) x 100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No animal died during the study period. One male animal of the 30 mg/kg bw group showed erosion and crust on the skin of the head during the dosing period; no females and no animal in the recovery group showed any changes in general conditions.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
For males and females, there were no significant differences in body weight between the control and each of the other administration groups. For males and females, there were no significant differences in food consumption between the control and each of the other administration groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no significant differences in the mean number of days for return of estrus in estrous cycle between the control and each of the other administration groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All animals copulated, and there were no significant differences in the number of pairing days until copulation between the control and each of the other administration groups. Additionally, all the copulated animals became impregnated, and there were no significant differences in the gestation index between the control and each of the other administration groups.
All the impregnated animals delivered liveborn pups. No abnormal delivery was seen in the animals. Abnormal lactation was found for 1 case in the 300 mg/kg bw group, which did not nurse pups on and after day 2 of lactation, and the pups were in a status of neglect.
There were no significant differences in the number of corpora lutea of pregnancy, number of implantation sites and implantation index between the control and each of the other administration groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Absolute testis weight in the 30 mg/kg bw group was significantly lower, and absolute epididymis weights in the 30 and 100 mg/kg bw groups were significantly lower. At the end of the recovery period, the 300 mg/kg bw group showed significantly higher values of absolute and relative epididymis weights than the control group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
In the examined organs/tissues of males and females, there were no findings likely to be caused by the administration of the test substance.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects were found at highest concentration tested

Results: F1 generation

Details on results (F1)

VIABILITY & CLINICAL SIGNS (OFFSPRING)
There were no significant differences in the number of pups born at birth, delivery index, number of liveborn pups, live birth index, viability index on day 0 of lactation and sex ratio between the control and each of the other administration groups. Moreover, no offspring showed abnormal appearance.

OBSERVATIONS DURING LACTATION
Apart from 1 case in the 300 mg/kg group showing abnormal lactation, there was no case of low viability index concerning offspring. Since the offspring in this case could be fed little milk, their body temperatures and activities decreased; and they died with the exception of one. Additionally, for 1 pup in the control group, a deficiency of hindlimbs (cut-off limbs) was found on day 4 of lactation. Moreover, there were no significant differences in the viability index on day 4 of lactation and sex ratio between the control and each of the other administration groups.

BODY WEIGHT (OFFSPRING)
There were no significant differences in body weight of offspring on days 0 and 4 of lactation between the control and each of the other administration groups.

GROSS PATHOLOGY & HISTOPATHOLOGY (OFFSPRING)
At necropsy on day 4 of lactation, 1 offspring in the 30 mg/kg group showed white spots on the liver, and 1 case in the control group exhibited a deficiency of hindlimbs (cut-off limbs). There was no other case showing abnormal findings, including the offspring which died in the middle of the lactation period.

Effect levels (F1)

Dose descriptor:

NOAEL

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.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No effects of the compound were observed on the reproductive performances, such as the estrous cycle, copulation, fertility, delivery and lactation. No effects of the compound were observed on the implantation, the number of pups, sex ratio of pups, viability of the pups during 4 days of lactation. No external abnormalities or macroscopic findings were detected in any pup. Therefore, the NOAEL for parental and F1 animals was found to be 300 mg/kg bw, the highest dose tested in this study.