2-(2-oxo-5-(1,1,3,3-tetramethylbutyl)-2,3-dihydro-1-benzofuran-3-yl)-4-(1,1,3,3-tetramethylbutyl)phenyl acetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

OECD guideline study, GLP-compliant. Vehicle control and low dose females were dosed inadvertently with formulation from another study on Friday 25 May 2007 (week 16 of study). Group 1 females received a vehicle (Milli-Q water) which was used in many other (reproduction) studies with no problems. Group 2 females were dosed with the test substance (a phthalate) of study 481747 (a two generation study) for one day at a dose level of 60 mg/kg. In this two-generation study, male and female Wistar rats were exposed at 0, 60, 200 and 600 mg/kg body weight/day for the first generation (second generation ongoing). No reproductive, breeding or developmental toxicity was observed up to 600 mg/kg. No parental toxicity was observed at 60 mg/kg/day when dosed for at least 14 weeks. For the above reasons, we consider the unfortunate dosing error of negligible impact on the scientific validity of project 484493.

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Version / remarks:

adopted on 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302/EEC, "One-Generation Reproductive Toxicity Test", 1988

GLP compliance:

yes

Remarks:

NOTOX B.V., Hambakenwetering 7, 5231 DD ‘s-Hertogenbosch, The Netherlands

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Males: Charles River Deutschland, Sulzfeld, Germany.
Females: Charles River Laboratories, Wilmington, MA, USA.
- Age at study initiation: Males 5-6 weeks and females 13-14 weeks.
- Housing:
Pre-mating: animals were housed in groups of 4 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (Mill type, height 18 cm).
Post-mating: Males were housed in groups of 4 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). Females were individually housed in Macrolon cages (Mill type, height 18 cm).
Lactation: Offspring was kept with the dam until termination.
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: ad libitum, tap water
- Acclimation period: 5 days (males) or 4 days (females) prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): temperature of 21 ± 3°C (measured range: 17.9 -24.1°C)
- Humidity: 30 - 70% (actual range: 34 - 96%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES:
Males: 31.01. - 11.06.2007
Females: 29.03. - 11.06.2007

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
From start of the study until 25 February 2007, formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenized to a visually acceptable level. From 26 February 2007 until end of the study, the formulations were prepared weekly. Adjustment was made for specific gravity of the vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX
- Amount of vehicle: 5 ml/kg body weight

Details on mating procedure:

- M/F ratio per cage: one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates)
- Length of cohabitation: maximum of 15 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of a intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility for an additional 5 days.
- Further matings after two unsuccessful attempts: no
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Accuracy, homogeneity and stability were determined for formulations prepared for use in Week 1, Week 8, Week 16 and Week 18 of study.

Duration of treatment / exposure:

F0-males: A minimum of 70 days prior to mating and continuing until necropsy.
F0-females: A minimum of 14 days prior to mating and continuing until necropsy.
F1-generation (F0-offspring): The F1-generation was potentially exposed to the test substance in utero and through nursing during lactation.

Frequency of treatment:

Once daily for 7 days per week. Animals were dosed up to the day prior to scheduled necropsy.

Details on study schedule:

- Selection of parents from F1 generation when pups were 4 days of age.
- Age at mating of the mated animals in the study: 16 -17 weeks

Remarks:

Doses / Concentrations:
0, 50, 150 or 1000 mg/kg bw/d
Basis:
nominal conc.

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on results of a 28-day toxicity study (NOTOX Project 264419). The test substance was chemically identical to the used substance in the present study. During the 28-day study animals were treated at 0, 50, 150 or 1000 mg/kg bw/d. No toxicity was observed up to 1000 mg/kg bw/d. From this information the same dose levels were selected.

Positive control:

Not applicable.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The time of death was recorded as precisely as possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
At least once daily, detailed clinical observations were made in all animals. The time of onset, degree and duration were recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 of gestation, and during lactation on Days 1, 4, 7,14 and 21. In order to monitor the health status, female 155 was weighed on 5 April 2007 (Day 4 of study).

FOOD CONSUMPTION AND COMPOUND INTAKE:
Weekly, for males and females. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and after delivery on Days 1, 4, 7, 14 and 21 post-partum.

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

Parameters examined in all male parental generations: testis weight, epididymis weight, prostate weight, pituatary weight, seminal vesicles weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
To reduce variability among the litters, on Day 4 of lactation, eight pups from each litter of equal sex distribution (if possible) were randomly selected. For litters consisting of fewer than eight pups, adjustments for litter size were not performed.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
Mortality/ viability
The numbers of live and dead pups at the First Litter Check (=check at day 1 of lactation) and daily thereafter were determined.

Clinical signs
At least once daily, detailed clinical observations were made in all animals.

Body weights
Live pups were weighed during lactation on days 1, 4, 7, 14 and 21.

Sex
Was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

Postmortem examinations (parental animals):

SACRIFICE
All animals surviving to the end of the observation period and all moribund animals were anaesthetised using iso-flurane and subsequently exsanguinated. Males were killed as soon as possible after delivery of the litters. Females were killed on Day 21 postpartum or shortly thereafter.

GROSS NECROPSY
After sacrifice or death all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin: Cervix, Coagulation gland, epididymides, ovaries, pituitary gland, prostate gland, seminal vesicles, testes, uterus, vagina, all gross lesions.

The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
Epididymides, ovaries, pituitary (weighed when fixed for at least 24 hours), prostate (weighed when fixed for at least 24 hours), seminal vesicles, testes, uterus

The following slides were examined by a pathologist:
- The preserved organs and tissues of 10 selected animals/sex of Group 1 (Males 1-10 and Females 97-106) and Group 4 (Males 74, 76-83, 85 and Females 170, 172-179, 181).
- The preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all animals suspected of infertility (e.g. those that failed to mate, conceive, sire or deliver healthy offspring):
Group 1 Males 19, 21 and Female 117
Group 2 Males 26, 46 and Female 122
Group 3 Males 57, 59 and Female 153
Group 4 Males 73, 75, 84, 86 and Females 169, 171, 180, 182
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Postmortem examinations (offspring):

SACRIFICE
Pups, which were culled, were killed by decapitation. All remaining pups were sacrificed using an oxygen/carbon dioxide procedure.
Culling was performed on Day 4 of lactation or shortly thereafter. The remaining pups were killed at Day 21 post partum or shortly thereafter.

GROSS NECROPSY
Offspring found dead or killed before Day 14 of lactation was sexed and externally examined (if practically possible) with emphasis on developmental morphology. The stomach was examined for the presence of milk.
Offspring killed on or after Day 14 of lactation was sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs with emphasis on developmental morphology. Descriptions of all macroscopic abnormalities were recorded. If possible, defects or cause of death were evaluated. No pups or tissues were fixed.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate, was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

Percentage mating, fertility index, conception rate, gestation index, gestation duration.

Offspring viability indices:

Percentage live males at first litter check, percentage live females at first litter check, percentage of postnatal loss days 0-4 post partum, percentage of breeding loss day 5 until weaning, percentage live males at weaning, percentage live females at weaning, viability index, weaning index.

MORTALITY (PARENTAL ANIMALS)
No treatment related mortality occurred during the study period. Two females died before the end of the study period. One animal treated at 50 mg/kg (Female 140) was sacrificed due to prolapse of the uterus on Day 1 of lactation and one animal treated at 150 mg/kg (Female 155) was found dead on Day 6 of treatment. No cause of death could be established.

CLINICAL SIGNS (PARENTAL ANIMALS)
No treatment related clinical signs were noted.
The female of the low dose group (animal number 140) that was killed in extremis showed prolapse of the uterus. The female treated at 150 mg/kg (animal number 155) that was found dead on Day 6 of treatment showed hunched posture, piloerection and laboured respiration on Days 4 and 5 of treatment. Incidental findings consisted of broken tail apex, chromodacryorrhea, alopecia, clonic spasms, quick breathing and scabs. These findings are occasionally noted in rats of this age and strain that are housed and handled under the conditions in this study and are therefore considered to be of no toxicological significance.

BREEDING DATA
One female of the control group (female 103) showed total litter loss (13 pups) on Day 2 of lactation. All fifteen pups of female number 140 (low dose group) had to be killed on Day 1 of lactation as this dam was killed in extremis due to prolapse of the uterus. Because of these litter losses, postnatal loss was statistically significantly decreased and viability index was increased for mid and high dose group animals.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights and body weight gain of treated animals remained in the same range as controls over the complete study period.
Additional measurement of body weight was performed for female 155 on Day 4 due to bad health status. On Day 4, this female showed a body weight loss of 31 g (- 13%) when compared to Day 1. The statistically significantly increased body weight gain observed on a few occasions was not considered toxicologically relevant.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals. The statistically significantly increased values for absolute food consumption noted on a few occasions were not considered toxicologically relevant.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Not determined.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Not determined.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproduction parameters were unaffected by treatment up to 1000 mg/kg body weight/day. Percentage mating and gestation indices were 100% for all Groups. The fertility indices and conception rates were 95.8% for Group 1 and 2, 95.7% for Group 3 and 83.3% for Group 4. These values were considered within normal limits (historical control data range is 79.2% to 100%). Mating performance, fertility parameters, duration of gestation and number of dead and living pups at first litter check were similar for the control and treated groups.
There were a total of ten males and seven females suspected of infertility:
2 males and 1 female of the control group (animals 19, 21, and 117), 2 males and 1 female of the low dose group (animals 24, 46 and 122), 2 males and 1 female of the mid dose group (animals 57, 59 and 153) and 4 males and 4 females of the high dose group (animals 73, 75, 84, 86, 169, 171, 180 and 182). In male number 84 (1000 mg/kg bw/d) marked seminiferous atrophy (bilateral) in the testes was present and marked oligospermia (bilateral) was recorded in the epididymides. These findings were considered to be the cause of infertility for this animal. They were not considered to be related to treatment as these were within the normal range of background pathology encountered in Wistar rats of this age and strain.
In the remaining suspect infertile animals there were no findings in macroscopy or histopathology which could be attributed to the test item to account for infertility.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically significant changes were noted regarding organ weights and organ:body weight ratios.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Necropsy did not reveal any toxicologically relevant alterations. The female of the low dose group (animal number 140) that was killed in extremis showed prolapse of the uterus. The female treated at 150 mg/kg bw/d (animal number 155) that was found dead on Day 6 of treatment did not show any macroscopic abnormalities. Incidental findings included bent tail apex, pelvic dilation of the kidneys, alopecia, urinary stones, nodule at the prostate, seminal vesicles reduced in size, testes reduced in size, epididymides reduced in size, and nodule at the uterine adipose tissue. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment related distribution they were considered changes of no toxicological significance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Morphologic alterations in the reproductive organs indicative of toxicity at doses up to 1000 mg/kg bw/d were not in evidence.
In animal 37 (50 mg/kg bw/d) an abscess with extensive fibrosis was seen in the prostate, correlating with the nodule recorded in this organ at necropsy. This finding in the prostate and the remainder of the microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar rats of this age.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item-related effects up to and incl. the higher dose level of 1000 mg/kg bw/d.

PUP DEVELOPMENT
Development of pups was unaffected by treatment up to 1000 mg/kg body weight/day.

VIABILITY (OFFSPRING)
Breeding parameters were unaffected by treatment up to 1000 mg/kg bw/d. No toxicologically relevant findings were observed for postnatal loss, living pups on Day 4 post partum, breeding loss between Days 5-21 post partum, living pups on Day 21 post partum, viability index and weaning index.

CLINICAL SIGNS (OFFSPRING)
Incidental clinical symptoms consisted of small appearance, wound, scabs, cold, no milk, chromodacryorrhea, pale appearance, missing tail apex and alopecia. These findings are occasionally noted in rats of this age and strain that are housed and handled under the conditions in this study and are therefore considered to be of no toxicological significance.

BODY WEIGHT (OFFSPRING)
Body weights of treated groups remained in the same range as controls. The statistically significantly increased body weight for male pups of the high dose group on Day 7 of lactation, was not considered toxicologically relevant.

SEXUAL MATURATION (OFFSPRING)
Not determined

ORGAN WEIGHTS (OFFSPRING)
Not determined
GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of the pups revealed small appearance, constricted liver, autolysis, no milk, constricted spleen, hernia diaphragmatica of the liver, cannibalism and alopecia. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain. Findings of note were observed for two pups. One pup of the low dose group (litter 139 female pup 6) showed the left eye closed and scabs on Days 21 and 22 of lactation. Macroscopic examination on Day 22 of lactation revealed desiccation of the left eye. One pup of the high dose group (litter 178 female pup 13) showed an enlarged head on Day 21 of lactation; macroscopic examination revealed hydrocephalis. At this incidence, these findings were considered unrelated to treatment.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item-related effects up to and incl. the higher dose level of 1000 mg/kg bw/d.

Reproductive effects observed:

not specified

Chemical analyses of dose preparations

It was concluded that the formulations were prepared correctly. The accuracies of the formulations were generally in good agreement with target concentrations. All formulations were homogeneous (i.e. coefficient of variation) and stable for at least 10 days when stored at room temperature.

Conclusions:

Based on the results of this study, the definitive parental, reproduction, breeding and developmental No Observed Adverse Effect Level (NOAEL) for the test substance was established as 1000 mg/kg/day.

Executive summary:

The test substance was assessed in a one-generation reproduction toxicity study following guideline OECD No. 415 and in compliance with GLP. After acclimatization, male and female Wistar rats of the F0-generation were exposed by oral gavage to graduated doses of the test substance. The dose levels were 50, 150 and 1000 mg/kg body weight/day. Chemical analyses of formulations were conducted during the study to assess accuracy, homogeneity and stability (10 days at room temperature). Males were exposed for 99 or 100 days, i.e. 10 weeks prior to mating, during mating, and up to termination. Females were exposed for 57 to 63 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 20 days of lactation. F0-females were allowed to produce and rear a litter until Day 21 of lactation. On Day 4 of lactation litters were reduced in size to eight pups by random culling of F1-pups. All animals were subjected to daily clinical observation. Body weight and food consumption were measured on several occasions over the treatment period. At necropsy, macroscopic observations and organ weights were recorded. Histopathology of reproductive organs was performed. Reproduction parameters, breeding data and pup development were assessed. There were no changes for mortality, clinical signs, body weight, food consumption, macroscopic examination, organ weights, microscopic examination, reproduction, breeding data and pup development that were considered to be an effect of treatment. From the results presented in this report a definitive parental, reproduction, breeding and developmental No Observed Adverse Effect Level (NOAEL) for the test substance of at least 1000 mg/kg/day was established.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

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 GLP-compliant one-generation reproduction toxicity study was performed according to OECD guideline 415 in Wistar rats. Male and female rats of the F0-generation were exposed by oral gavage to graduated doses of the test substance. The dose levels were 50, 150 and 1000 mg/kg body weight/day. Males were exposed for 99 or 100 days, i.e. 10 weeks prior to mating, during mating, and up to termination. Females were exposed for 57 to 63 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 20 days of lactation. F0-females were allowed to produce and rear a litter until Day 21 of lactation. On Day 4 of lactation litters were reduced in size to eight pups by random culling of F1-pups. Accuracy, homogeneity and stability (10 days at room temperature) of formulations were demonstrated by analyses. There were no changes for mortality, clinical signs, body weight, food consumption, macroscopic examination, organ weights, microscopic examination, reproduction, breeding data and pup development that were considered to be an effect of treatment. From the results presented in this report a definitive parental, reproduction, breeding and developmental No Observed Adverse Effect Level (NOAEL) for TKA 40213 (CGP 2160) of at least 1000 mg/kg/day was established.

Short description of key information:
In an one-generation study (Notox, 2007) performed according to GLP principles and OECD guidelines, the definitive parental, reproduction, breeding and developmental No Observed Adverse Effect Level was established as being at least 1000 mg/kg/day.

Justification for selection of Effect on fertility via oral route:
GLP compliant guideline study

Effects on developmental toxicity

Description of key information

No data available on teratogenicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data is reliable and suitable for the purpose of classification under Directive 67/548/EEC. Based on the present data, classification for reproductive toxicity is not warranted under Directive 67/548/EEC.

 

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

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for reproductive toxicity is not warranted under Regulation (EC) No.1272/2008.