Phenol, heptyl derivs.

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2011-March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to OECD and US EPA guidelines and according to GLP principles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3550

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Phenol, heptyl derivs.

EC Number:

276-743-1

EC Name:

Phenol, heptyl derivs.

Cas Number:

72624-02-3

Molecular formula:

C13H20O

IUPAC Name:

4-(2-methylhexyl)phenol; 4-heptylphenol

Details on test material:

- Physical state: light brown, clear, viscous liquid
- Storage condition of test material: stored at room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI or Raleigh, NC
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Males: 331-391 g; Females: 211-291 g
- Fasting period before study: no
- Housing: until pairing: individually in stainless steel wire-mesh cage; paired for mating in male cage; after mating males were housed in suspended wire-mesh cages; females were transferred to plastic maternity cages with nesting material, ground corncob bedding.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet: ad libitum PMI Nutrition International, LLC Certified Rodent LabDiet 5002
- Water: ad libitum (reverse-osmosis-purified on-site drinking water)
- Acclimation period: minimum of 10 days for males and 15 days for females

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2-21.4
- Humidity (%): 42.9-48.8
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2 December 2011 To: 22 January 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance formulations were prepared approximately weekly for each dosage level, divided into daily aliquots and stored refrigerated. Test substance concentrations were 0, 4, 8, 16 and 32 mg/mL.
Dosage volume for all groups was 5 mL/kg.

VEHICLE
- Lot/batch no.: 2AD0465

Details on mating procedure:

- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug or the presence of sperm following a vaginal lavage referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually into plastic maternity cages with nesting material, ground corncob bedding.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were collected from the middle stratum of each dosing formulation (including control group) prepared during the first and the last week of dose administration. Analyses were conducted by means of a validated gas chromatography method using flame ionization detection. Mean concentrations ranged from 95.3% to 108% of target concentration.

Duration of treatment / exposure:

males: 14 daily doses prior to mating; throughout mating period for a total of 31 doses
females: 14 daily doses prior to pairing, dosed through lactation day 3 (females that delivered) or post-mating day 25 (females that failed to deliver) for a total of 39-51 doses

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 20, 40, 80, 160 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of the 28-day OECD 407 study, where a NOAEL of 150 mg/kg bw/day was observed (no significant effects observed). For the present screening study a top dose of 160 mg/kg bw/day was included to increase the probability for detecting some adverse effects, without risking the possibility of excess toxicity due to the longer duration.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- for moribundity and mortality, and for signs of toxicity 2 hours following dose administration. Females expected to deliver were observed for dystocia or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for males and weekly for females until evidence of copulation. Female body weights were recorded on GD 0, 4, 7, 11, 14, 17 and 20 and on lactation days 0, 1 and 4.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymides weight, seminal vesicle weight; microscopic examinations of testes, epididymides, seminal vesicles, coagulating glands and prostate gland.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
- all pups

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
litter size, 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

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following completion of the mating period.
- Maternal animals: All surviving animals on lactation day 4 (females that delivered) or on post-mating day 25 (females that failed to deliver)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including external surface, all orifices and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Moreover the number and location of corpora lutea and implantation sites were examined , and uteri were investigated for early implantation loss.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues according to guidelines were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 4 days of age and discarded.

Statistics:

Analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Parental mating, fertility, conception and copulation indices were analyzed using the Chi-square test with Yates' correction factor. A parametric one-way ANOVA to determine intergroup differences was applied to mean parental BW, BW changes and food consumption, offspring BW and BW changes, gestation length, numbers of implantation sites, number of pups born, live litter size on PND 0, corpora lutea, organ weights (absolute and relative) and pre-coital intervals. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Kruskal-Wallis nonparametric ANOVA was used to determine intergroup-differences for mean litter proportions of males at birth and postnatal survival. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn's test was used to compare the test substance-treated groups to the control group. Histopathological findings of treated groups were compared to the control group by the Fisher's Exact test.

Reproductive indices:

Mating, fertility, and copulation/conception indices were calculated as follows:

Male (Female)
Mating Index (%) = (No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant)/Total No. of Males (Females) Used for Mating) x 100

Male Fertility
Index (%) = (No. of Males Siring a Litter/Total No. of Males Used for Mating) x 100

Male Copulation Index (%) = (No. of Males Siring a Litter/No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy)) x 100

Female Fertility
Index (%) = (No. of Females with Confirmed Pregnancy/Total No. of Females Used for Mating) x 100

Female Conception Index (%) = (No. of Females with Confirmed Pregnancy/ No. of Females with Evidence of Mating (or Confirmed Pregnancy)) x 100

Offspring viability indices:

Litter parameters were defined as follows:

Mean Live Litter Size = Total No. of Viable Pups on PND 0/ No. of Litters with Viable Pups PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-selection) (% Per Litter) =
(Sum of (Viable Pups Per Litter on PND 0 or PND 4 [Pre-selection]/No. of Pups Born Per Litter)/No. of Litters Per Group) x 100

Postnatal Survival for All
Other Intervals (% Per Litter) = (Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)/No. of Litters Per Group) x 100

Where N= PND 0-1 and 1-4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

non-dose related salivation

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slightly decreased food intake and BW gain in males only

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slightly decreased food intake and BW gain in males only

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

male liver and kidneys

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
Salivation prior to dosing and clear material around nose and mouth are considered test substance related but not to be adverse.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
High dose males showed slightly lower, statistically significant during days 13-20 and 0-31, mean BW gain on day 31 (-6.3%) compared to controls.

ORGAN WEIGHTS (PARENTAL ANIMALS):
Mean (absolute and relative) liver weights were statistically significant increased in high dose females by 16 and 19.8%, respectively. In high dose males relative liver weight was statistically significant increased (17%) and relative kidney weight was increased by 12.7%. These observations are considered non-adverse.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In high dose males increased hepatocellular vacuolation (minimal and mild) were observed; kidneys of high dose males showed an increase in basophilic tubules and an increase in minimal tubular dilatation. These observations are considered non-adverse.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects slightly lower bw gain in high dose males.

2

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 160 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

OTHER FINDINGS (OFFSPRING):
Lower mean number of pups born (11.9 and 11.3 pups per dam) were observed in the 80 and 160 mg/kg bw/day groups, respectively (control group 14.5 pups per dam). However, the results from the 80 mg/kg bw/day group are comparable to the historical minimum values observed in WIL historical control data, 11.7 pups per dam. Furthermore, the lower mean number of pups born in the high dose group were primarily attributed to one female (single pup born). Excluding this female , -mean number of pups born was 12.2, which is well within WIL historical data ranges. The observed lower mean values for pups born in the 80 and 160 mg/kg bw/day groups are considered therefore not toxicologiccally relevant or test-substance related.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

>= 160 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental toxicity was observed up to the highest dose level tested (160 mg/kg bw/day).

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Treatment with test item by oral gavage in male and female Sprague-Dawley rats at dose levels of 20, 40, 80 and 160 mg/kg bw/day according to EU/EPA test guidelines resulted in slightly lower body weight gain in high dose male rats. Based on these observations in high dose animals, the NOAEL for parental systemic toxicity is 80 mg/kg bw/day. No reproduction and developmental toxicity was observed for treatment up to 160 mg/kg bw/day. A reproduction and developmental NOAEL of at least 160 mg/kg bw/day was derived.

Executive summary:

Test Guidance

According to OECD 421 and US EPA OPPTS 870.3550 guidelines

Method and materials

Test item was administrated by daily oral gavage to male and female Sprague-Dawley rats at dose levels of 20, 40, 80 and 160 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating and up to termination (total of 31 doses). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and during 3 days of lactation (39 -51 doses).

Results

Analysis demonstrated mean concentration ranges from 95.3% to 108% of target concentrations.

Parental toxicity was observed at the high dose only, and consisted of slightly lower body weight gain in high dose male rats at 160 mg/kg bw/day. The observed statistically significant increased relative liver weights in males and females, and increased absolute liver weights in females, together with increased hepatocellular vacuolation and increased minimal tubular dilation in kidneys observed in high dose males are considered non-adverse. Based on these observations in high dose animals, the NOAEL for parental systemic toxicity is 80 mg/kg bw/day. Maternal toxicity was at least 160 mg/kg bw/day, as no adverse effects were observed in dams.

Conclusions

No reproduction and developmental toxicity was observed for treatment up to 160 mg/kg bw/day. A reproduction and developmental NOAEL of at least 160 mg/kg bw/day was derived.