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Effects on fertility

Description of key information

read-across CAS 2440-22-4: OECD 422, rat, gavage study - NOAEL = 300 mg/kg bw (parental and F1)

Link to relevant study records
Reference
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.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
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
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals 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
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
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
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
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.

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
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.
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.
Reproductive effects observed:
not specified
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.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
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

There are no studies available assessing reproductive toxicity of 2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol (CAS 3147-75-9). Read-across from the structurally related substance 2-(2’-Hydroxy-5’-methylphenyl) benzotriazole (CAS 2440-22-4) was applied.

The read-across justification including a data matrix are part of the chemical safety report.

A combined repeated dose toxicity/reproduction toxicity study with gavage application of 2-(2’-Hydroxy-5’-methylphenyl) benzotriazole (CAS 2440-22-4) was performed in 2006 (JBRC, 2012 – new translation). The GLP compliant study followed OECD testing guideline 422. Olive oil was used as vehicle. The test substance was applied by gavage to male animals for 42 days (14 days prior to, 14 days during and 14 days after mating) and to female animals for up to 53 days (14 days prior to mating up until day 4 of lactation) at concentrations of 30, 100 and 300 mg/kg bw. The choice of concentrations applied was based on the systemic toxicity to parental animals. The NOAEL for systemic toxicity was found to be 30 mg/kg bw for male and female animals.

No effects of the test substance were observed on the reproductive performances, such as the estrous cycle, copulation, fertility, delivery and lactation. Additionally, no effects were observed for the offspring: implantation, number of pups, sex ratio, weight and viability up to day 4 of lactation. No external abnormalities or macroscopic findings were detected in any of the pups.

Overall, due to no effects seen, the NOAEL for the reproductive developmental toxicity was found to be 300 mg/kg bw, the highest concentration tested in this study. 




Effects on developmental toxicity

Description of key information
read-across CAS 2440-22-4: OECD 414, rat, gavage study - NOAEL = 1000 mg/kg bw 
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
not indicated
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.
Qualifier:
equivalent or similar to
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:
CAS 2440-22-4
Species:
rat
Strain:
Sprague-Dawley
Details on test animals 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.
Remarks:
Doses / Concentrations:

Basis:
actual ingested
150 mg/kg bw
Remarks:
Doses / Concentrations:

Basis:
actual ingested
500 mg/kg bw
Remarks:
Doses / Concentrations:

Basis:
actual ingested
1,000 mg/kg bw
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
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)
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The substance causes no teratogenicity, embryotoxicity, developmental toxicity and maternal toxicity in rats.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
Acceptable quality although not conducted under GLP
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

There are no studies available assessing reproductive toxicity of 2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol (CAS 3147-75-9). Therefore, read-across from the structurally related substance 2-(2’-Hydroxy-5’-methylphenyl) benzotriazole (CAS 2440-22-4) was applied.

The read-across substance did not induce adverse effects on embryo-fetal development up to and including the high dose of 1000 mg/kg/day.

Justification for classification or non-classification

Based on the available read-across data and according to CLP Regulation (EU) 1272/2008 and Directive 67/548/EEC, respectively,

classification of 2 -(2H-Benzotriazol-2 -yl)-4 -(1,1,3,3 -tetramethylbutyl)phenol is not warranted.