Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-11-04 to 2014-06-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Cross-referenceopen allclose all
Reason / purpose:
read-across: supporting information
Reason / purpose:
reference to same study
Remarks:
Sections 7.5.1. and 7.8.2.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
deviations were considered not to have any influence on the validity and integrity of the study results
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Rosin, hydrogenated
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: UVCB
- Physical state: Solid, glassy, amber granules
- Analytical purity: 100%
- Purity/Concentration for Formulation: 100%
- Lot/batch No.: MR043
- Expiration date of the lot/batch: 09-Jul-2014
- Stability of the test item in Feed Preparation: At least 28 days if stored at room temperature (20 ± 5 °C) in the dark.
- Storage condition of test material: At least 28 days if stored at room temperature (20 ± 5 °C) in the dark.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Eastman Chemical Company; Batch No. MR043
- Expiration date of the lot/batch: 2014-07-09
- Purity test date:2013-07-13

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Bulk sample was stored frozen (-20 ± 5 °C) under nitrogen in the dark. For temperature adjustment, working samples were kept under nitrogen at room temperature (20 ± 3 °C) for a maximum of 3 hours, prior to feed preparation.
- Stability under test conditions: At least 28 days if stored at room temperature (20 ± 5 °C) in the dark.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Solid, glassy, amber granules

Test animals

Species:
rat
Strain:
other: RccHan TM : WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst / Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 12 wks
- Weight at study initiation: (P) Males: 311 - 377 g; Females:188 - 217 g
- Fasting period before study: Not specified
- Housing: During acclimatization and pre-pairing in groups of up to three animals by sexin Makrolon type-4 cages, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles. During pairing females were housed with males (1:1) in Makrolon type-3. Following the pairing period males were re-housed in their original pre-pairing cages and females were individually housed in Makrolon type-3 cages. All cages were provided with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK and ISO-BLOX from Harlan Laboratories B.V. / Netherlands). In addition a wood stick was provided (batch nos. 02105130819, 77, 6960C-100267 and 132211).
- Diet (e.g. ad libitum): Microgranulated standard Harlan Teklad 2018C (batch no. 41/13) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitumin water bottles
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

IN-LIFE DATES: From: 2013-10-31 To: 2014-01-01

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were prepared at intervals not exceeding 28 days using the test item as supplied by the supplier.
- Mixing appropriate amounts with (Type of food): A portion of Rosin, Hydrogenated was ground to powder using an electrical grinder, weighed into a tared glass beaker on a suitable precision balance, and mixed with microgranulated feed separately for each dose group. No heat was used during diet preparation without consultation with, and agreement from, the Sponsor. Control feed for the animals of group 1 was prepared similarly, but without test item.
- Storage temperature of food: Feed preparations were stored at room temperature (20 ± 5 °C), protected from light in metal containers until use
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation was observed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for analyses of test item, its content and homogeneity in the feed were drawn at the start of the pre-pairing period, and at the end of the gestation/start oflactation (i.e. two occasions). Stability of the test item in the feed for 28 days at room temperature was determined at the start of the pre-pairing period (i.e. one occasion). For assessment of content and homogeneity, samples of approximately 100 g were collected from each the top, middle and bottom of every dietary admixture of the respective diet preparation (from group 1 admixtures only one sample was drawn from the middle of the preparation). Samples for assessment of content and homogeneitywere collected on the day of preparation. For assessment of stability, samples of approximately 100 g were drawn from the middle of every dietary admixture on the day of preparation and stored at room temperature for 28 days. After preparation the samples were delivered to the analytical department at ambient temperature and analyzed immediately or stored there frozen (at -20 ± 5 °C) until analysis. The samples were analyzed by HPLC coupled toan LC/MS detector following an analytical procedure developed at Harlan Laboratories. The test item was usedas the analytical standard. Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Itingen / Switzerland. The samples were discarded after finalization of the report without any further note.
Duration of treatment / exposure:
Males: 43 days (during 14 days pre-pairing period, 15 days pairing period and 14 days postpairing period).
Females: Minimum 5 weeks (14 days pre-pairing period, up to 15 days pairing period, approximately 21 days of gestation period and lactation period to day 4 post partum).
Frequency of treatment:
Continuous (ad libitum)
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control (Group 1)
Dose / conc.:
1 000 ppm
Remarks:
Low Concentration (Group 2)
Dose / conc.:
3 000 ppm
Remarks:
Intermediate Concentration (Group 3)
Dose / conc.:
10 000 ppm
Remarks:
High Concentration (Group 4)
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selectedbased on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories study no. D80948, using concentrations in feed of 5000, 10000 and 20000 ppm and resulting in adverse reduction in food consumption and body weight loss at the high-dose level and less pronounced effects on food consumption and body weights at the mid-dose level.

- Rationale for animal assignment (if not random): Performed after at least three days of acclimatization using a computer-generatedrandom algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar
mean body weights in all groups.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males: Once prior to the first administration of the test item and weekly thereafter.
Females: Once prior to the first administration of the test item, weekly during the pre-pairing and pairing and on days 0, 6, 13 and 20 post coitum.

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Once during acclimatization for randomization (not reported), at three-day intervals during pre-pairing and post-pairing periods and weekly during pairing period.
Females: Once during acclimatization for randomization (not reported), at three-day intervals during pre-pairing period, weekly during pairing period and on the following days: 0, 7, 14 and 20 post coitumand 1 and 4 post
partum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Males: At three-day intervals during pre-pairing and post-pairing.
Females: At three-day intervals during pre-pairing and for periods: days 0 - 7, 7 - 14 and 14 - 20 post coitumand 1 - 4 post partum. No food consumption was recorded during the pairing period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily water intake was measured gravimetrically during the entire studywith exception for acclimatization and pairing periods.

OTHER:

- Functional Observational Battery: Five males shortly before the scheduled sacrifice and five females on day 3 post partum were subjected to tests battery

- Viability/Mortality: Twice Daily

- Hematology and Clinical Biohemistry: Blood samples were obtained on the day before or on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Tables 2 and 3 provide data on hematological and clinical biochemistry parameters.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other: righting reflex]

GROSS EXAMINATION OF DEAD PUPS: yes, All pups, except those excessively cannibalized, were examined macroscopically for any structural changes,either at the scheduled necropsy or during the study if death occurred.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving males were sacrificed on the day after completion of the treatment for 43 days, when they were no longer necessary for the assessment of reproductive performance.
- Maternal animals: All surviving dams were sacrificed on day 5 post partum.

GROSS NECROPSY
- All animals surviving to the end of the observation period were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguinations. All parent animals, except those excessively cannibalized, were examined macroscopically for any structural changes,either at the scheduled necropsy or during the study if death occurred. For the parent animals, special attention was directed at the organs of the reproductive system. The uteri of all dams were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites and the number of implantation sites was noted. The number of corpora luteain each ovary was recorded for all pregnant females.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the scheduled sacrifice, organsfrom P parental animals were weighted and preserved as listed in tables 4.and 5 Organs were trimmed from any adherent tissue, and their wet weight taken with exception for thyroids and pituitary which weights were taken after fixation. If not indicated otherwise in the table, organswere preserved in neutral phosphate buffered 4% formaldehyde solution
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on day 4 post partum.

GROSS NECROPSY
- All pups, except those excessively cannibalized, were examined macroscopically for any structural changes,either at the scheduled necropsy or during the study if death occurred.
Statistics:
The following statistical methods will be used to analyze food consumption, water consumption, body weights, clinical laboratory data, organ weights, macroscopical findings, grip strength, reproduction data and righting reflex:
• Means and standard deviationsof various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
1) Mating Performance and Fertility
2) Corpora Lutea Count
3) Duration of Gestation
4) Implantation Rate and Post-Implantation Loss
5) Post natal loss
Offspring viability indices:
1) First Litter Check: Offspring were examined as soon as possible after completion of delivery for litter size, number of live and still births, and any gross abnormalities.
2) Viability / Mortality: Daily.
3) Clinical Signs: Daily observations for any abnormal findings.
4) Sex: On days 0 (if possible), 1 and 4 post partum.
5) Body Weights: On days 0 (if possible), 1 and 4 post partum.
6) Surface righting reflex: All pups were subjected to surface righting reflex on day 1 post partumas described in Section

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related clinical signs were observed in males or females in any group. A missing incisor and an overgrowing, malpositioned incisor recorded in females in group 3 were confirmed during detailed clinical observations. These findings were
considered not to be related to the treatment with the test item due to isolated occurrence.
Mortality:
no mortality observed
Description (incidence):
All animals survived until the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males - Pre-Pairing, Pairing and Post-Pairing Periods
Treatment with the test item caused a reduction in body weight gain resulting in reduced body weights in group 4. Mean body weight gain in order of ascending dose levels was 12.8%, 12.2%, 12.0% and 3.7% during the pre-pairing period, 9.2%, 6.1%, 7.4% and 8.6% during the pairing period and 4.0%, 4.7%, 5.2% and 4.2%during the post-pairing period. Body weight loss up to 3.8% was noted in group 4 on day 4 of the pre-pairing period, followed by statistically significantly reduced body weight gain recorded from day 4 until the end of this period. During the pairing period, body weight gainwas slightly, not statistically significantly lower and during the post-pairing period similar to the respective control values. Throughout treatment, the mean body weight was no less that 88% of the control value at the same time point. Statistically significant differences from control were occasionally recorded in groups 2 and 3, but overall values of body weights and body weight gain in these groups were within the control range and not considered to be affected by the treatment.

Females - Pre-Pairing, Gestation and Lactation Periods
Treatment with the test item caused a reduction in body weight gain resulting in reduced body weights in group 4. Mean body weight gain in order of ascending dose levels was 8.8%, 8.0%, 7.7% and -0.5% during the pre-pairing period, 53.3%, 50.4%, 56.4% and 43.4% during the gestation period and 7.4%, 7.0%, 9.0% and 5.5% during the lactation period. Body weight loss up to 3.7% was recorded in group 4 on day 4 of the pre-pairing period and reduced body weight gain until the completion of the study. This reduction was statistically significant during the entire pre-pairing period and on day 20 of the gestation period. Consequently, statistically significantly reduced body weights wererecorded from day 4 of the pre-pairing period until the completion of the study. During the pre-pairing period and until day 14 of gestation the reduction in mean body weightwas within 10% of the control value at the same time point, thereafter the reduction in body weight was within15% of the control value. Statistically significant differences from control were occasionally recorded in groups 2 and 3, but overall values of body weights and body weight gain in these groups were within the control range and not considered to be affected by the treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males - Pre- and Post-Pairing Periods
Treatment with the test item caused a reversible reduction in food consumption in group 4. In order of ascending dose levels, mean food consumption was 23.3, 21.9, 22.6 and 18.1 g/animal/day during the pre-pairing period and 25.6, 25.6, 24.9 and 23.3 g/animal/day during the post-pairing period. The reduction in food consumption in group 4 was statistically significant from day 1 to 13 of the pre-pairing period but exceeded the control value from day 13 to 14 of this period, although this was not statistically significant. During the post-pairing period, food consumption in this group was not statistically significantly different from control. In groups 2 and 3, food consumption was similar tothat in the control group during the entire study.

Females - Pre-Pairing, Gestation and Lactation Periods
Treatment with the test item caused a reversible reduction in food consumption in group 4. In order of ascending dose levels, mean food consumption was 15.2, 15.5, 15.7 and 12.0 g/animal/day during the pre-pairing period, 22.1, 22.1, 22.3 and 18.9 g/animal/day during the gestation period and 24.4, 25.7, 29.5 and 24.3 g/animal/day during the lactation period. The reduction in food consumption in group 4 was statistically significant from day 1 to 10 of the pre-pairing period. During the remaining pre-pairing period food consumption in this group was slightly lower or similar tothe control values. During the gestation period food consumption was lower than that of the control group, but only reached statistical significance on days 0 to 7 and 14 to 20 post partum. During the lactation period food consumption was similar to that in the control group. In groups 2 and 3, food consumption was similar tothat in the control group during the entire study.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Males - Pre- and Post-Pairing Periods
Treatment with the test item caused a reversiblereduction in food conversion efficiency in group 4. In order of ascending dose levels, mean food conversion efficiency was 0.052, 0.056, 0.056 and 0.049 g bw/g food/animal/day during the pre-pairing period and 0.018, 0.022, 0.022 and 0.022 g bw/g food/animal/day during the post-pairing period. In group 4, a statisticallysignificant reduction in food conversion efficiency was noted from day 1 to 7 of the pre-pairing period. Afterwards, food conversion efficiency recovered and was statistically significantly higher than the control value from day 13 to 14 of the pre-pairing period and similar to the control values during the post-pairing period. In groups 2 and 3, food conversion efficiency was similar or slightly higher than in the control group during the entire study.

Females - Pre-Pairing, Gestation and Lactation Periods
Treatment with the test item caused a reduction in food conversion efficiency in group 4. In order of ascending dose levels, mean food conversion efficiency was 0.029, 0.031, 0.029 and 0.014 g bw/g food/animal/dayduring the pre-pairing period, 0.286, 0.263, 0.286 and 0.241 g bw/g food/animal/day during the gestation period and 0.255, 0.198, 0.261 and 0.193 g bw/g food/animal/day during the lactation period. in group 4, the reduction in food conversion efficiency was statistically significant from day 1 to 4 and 7 to 10 of the pre-pairing period and from day 14 to 20 of the gestation period. No statistically significant changes in food conversion efficiency were noted during the remaining
study period. No test item-related effects on food conversion efficiency were observed in groups 2 and 3.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No effects on water consumption were observed in males or females in any group.
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related effects on hematology parameters were noted in males or females at any dose level.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males - Treatment with the test item caused an increasein activity of alanine aminotransferase and alkaline phosphatase in group 4. Mean activity of alanine aminotransferase was 60.7 U/L in group 4 versus 31.7 U/L in the control group whereas mean activity of alkaline phosphatase was 119.8 U/L in group 4 versus 67.2 U/L in the control group. Remaining statistically significant changes were: higher concentration of urea in group 4, lower concentration of bilirubin in group 2, higher concentration of triglicerides in group 2, higher concentration of sodium in group 3, higher concentration of potassium in group 4, higher concentration of calcium in all treated groups and higher concentration of albumin in group 4. These differences were minor, affected values remained in the range or close to the historical controls and several changes did not follow a dose dependency and therefore they were considered not to be related to the treatment with the test item.

Females - No test item-related effects on biochemistry parameters were noted in females at any dose level.
Following statistically significant changes were noted: higher concentration of bilirubin in group 4, lower activity of aspartate aminotransferase in group 4, higher concentration of chloride in group 4, lower concentration of albumin in group 2. All these changes were only minor, values remained close to or in the range of historical controlsand therefore they were considered not to be related to the treatment with the test item.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional Observational Battery: No test item-related findings were recorded during functional observational battery in males or females in any group.

Locomotor Activity: Locomotor activity was considered not to be affected by the treatment with the test item.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test-item related findings were observed in the adrenal glands of males in groups 3 and 4 and females in groups 2, 3 and 4, in the liver and pituitary of males in group 4. In the adrenal glands, minimal hypertrophy of the zona glomerulosa was recorded with an overall dose-related incidence: the incidence in males of groups 1, 2, 3, and 4 was 0/12, 0/12, 2/12, and 4/12 respectively; the incidence in females of groups 1, 2, 3, and 4 was 0/12, 3/12, 3/12, and 7/12 respectively. The change was characterized by diffusehypertrophy of the zona glomerulosa associated with vacuolisation of the zona glomerulosacells. In group 4 in males, there were minimal to slight hepatocellular hypertrophy in the liver and minimal multifocal cell hypertrophy in the pituitary. In the liver, the enlarged hepatocytes displayed a “ground glass appearance”. In the pituitary, the finding was characterized by an increased number of scattered enlarged pale eosinophilic cellswithin the pars distalis.
Compared to the normally expected, decreased cortical lymphocytes were observed in the thymus of some rats in all female groups including control group, the incidence/severity being however slightly higher in females in group 4, most likely related to non-specific stress.

Reproductive function / performance (P0)

Reproductive performance:
no effects observed
Description (incidence and severity):
1) Mating Performance and Fertility:
With exception for two females in group 3 (nos. 73 and 83), in all females mating was observed within the first pairing period. Precoital time was unaffected by the treatment with the test item. Mean (median) precoital times were 2.9 (3), 3.1 (3), 2.3 (3) and 2.8 (3) days in groups 1, 2, 3 and 4, respectively. For female nos. 73 and 83, no mating was observed during the first pairing period and therefore a second pairing period with another male was initiated. Female no. 83 mated successfully during the additional pairing. In female no. 73, body weight gain indicating pregnancy was observed and therefore the second pairing period had been stopped. Female no. 73 was pregnant and gave birth. Based on the birth date, the first pairing partner (male no. 25) was successful in mating. Consequently, percentage mating (number of animals mated as a percentage of females paired) was for males 92% in group 3 and 100% in the remaining groups and for females 100% in all groups.

With the exception of one female in each group 1, 3 and 4 (nos. 50, 76 and 85, mated with male nos. 2, 28 and 37, respectively), all mated females were pregnant and gavebirth to living pups. The gestation index (number of females with living pups as a percentage of females pregnant) was 100% in all groups whereas fertility index (number of females achieving pregnancy as a percentage of females paired) and conception rate (number of females achieving pregnancy as a percentage of females mated) were 91.7%, 100%, 91.7% and 91.7% in groups 1, 2, 3 and 4, respectively. The number of males producing offspring as a percentage of males paired was 91%, 92%, 83% and 91% whereas the number of males producing offspring as a percentage of males mated was 91%, 92%, 91% and 91% in order of ascending dose levels.

2) Corpora Lutea Count:
Mean number of corpora luteaper dam was 15.6, 13.6, 15.0 and 12.5 in groups 1, 2, 3 and 4, respectively. In group 4, mean number of corpora lutea was statistically significantly reduced if compared to the control value. This reduction was considered to be related to the treatment with the test item but not adverse.

3) Duration of Gestation: Duration of gestation was unaffected by exposure tothe test item.

4) Implantation Rate and Post-Implantation Loss: Mean number of implantations per dam and post-implantation loss were considered not to be affected by the treatment with the test item.

5) Litter Size at First Litter Check: Mean number of pups per dam at first litter check was 11.3, 9.8, 13.1 and 10.1 in groups 1, 2, 3 and 4, respectively. Number of pups per dam was slightly lower in group 4 if compared to the control group. The difference was however not statistically significant and value at the high dose level was in the range of historical controls, therefore it was considered not to be related to the treatment with the test item

6) Postnatal Loss Days 0 - 4 Post Partum: Post natal loss was not affected by the treatment with the test item at any dose level.

Details on results (P0)

There were no test item related microscopic findings in the reproductive organs, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries. There were no test item related microscopic findings in the males or females suspected of reduced fertility. Moderate diffuse vacuolation in the ovaries, minimal squamous metaplasia in the uterus and mucosal parakeratosis in the vagina were recorded in female no. 76. Owing to their isolated occurrence in the mid-dose group only, these findings were considered to be unrelated to treatment.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 000 ppm
System:
endocrine system
Organ:
adrenal glands
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related abnormal findings were noted in pups at first litter check or during lactation in any group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item resulted in slightly reduced pup body weights and body weight gain in group 4. Mean body weights of pups was 6.34, 6.26, 5.79 and 5.65 g on day 1 post partumand 9.14, 9.50, 8.21 and 7.76 g on day 4 post partum, whereas body weight gain was 42.71%, 51.59%, 40.84% and 37.03% during this period. In group 4, lower body weights were recorded on days 1 and 4 post partumwith statistical significance for female pup body weights on day 1 post partum. Also body weight gain of pups was lower in this group. Although most of the differences were not statistically significant, values of body weights were lower than historical controls and therefore changes in body weights were considered to be related to the treatment with the test item.
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex Ratios: Sex ratios at first litter check and on day 4 post partumwere unaffected by the exposure to the test item.

Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related findings were noted during necropsy of pups in any group.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Righting Reflex: No effects on righting reflex were observed in pups at any dose level.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the histopathological changes observed in adrenal glands, no NOEL (No Observed Adverse Effect Level) for systemic toxicity was established for females whereas for males it was at the dose level of 1000 ppm. The NOAEL (No Observed Adverse Effect Level) was established at 3000 ppm. For reproduction and developmental toxicity, the NOEL was established at the dose level of 3000 ppm whereas the NOAEL was at 10000 ppm, the highest dose level used in the study.
Executive summary:

In a key combined repeated dose, reproductive/developmental toxicity study, the test material (Rosin, hydrogenated; CAS# 65997-06-0) was continuously administered in feed to rats (12/sex/concentration) at concentrations of 0, 1000, 3000, and 10000 ppm. The test material was administered to male rats for 43 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

Test material-related effects were observed in males and females in group 4 (10000 ppm) whereas in groups 2 (1000 ppm) and 3 (3000 ppm) no signs of general toxicity were noted in either gender. Microscopic examination of selected organs revealed pathological changes in groups 2, 3 and 4.

 

In group 4, reduction in food consumption and food conversion efficiency resulting in body weight loss, reduced body weight gain and significantly reduced body weights were observed in both genders. The effect on food consumption was considered to most probably not be a sign of toxic potential but to result from unpalatability of the test item. Food consumption recovered during the study and therefore the effect was considered not to be adverse. Although body

weight gain increased and was similar to the control values after the initial reduction, body weights remained lower than the control group values during the entire study period in males and females. The persistent reduction in body weights was considered to be adverse.

 

An increase in activities of alanine aminotransferase and alkaline phosphatase were measured in the blood of males in group 4. This finding was considered to be indicative of liver cell injury and therefore adverse.

 

Increased liver weights were recorded at necropsy in males in group 4. This finding was correlated histologically with hepatocellular hypertrophy. Increased liver weights and hepatocellular hypertrophy are suggestive of an adaptative response to mixed function oxidase induction. In the pituitary, enlarged vacuolated cells were observed. This change may reflect hypertrophy of thyroid-stimulating hormone-producing cells (thyrotrophs), a common finding following

administration of liver enzyme inducers where the underlying mechanism is considered to be increased hepatic clearance of thyroid hormones followed by a compensatory increase in the pituitary secretion of TSH. Further, minimal hypertrophy of zona glomerulosa was observed during microscopic examinations in the adrenal glands of males in groups 3 and 4 and of females in groups 2, 3 and 4. The pathogenesis of this change is uncertain. The zona glomerulosa is the site of synthesis of aldosterone a mineralocorticoid which is mainly involved in the control of salt and water balance in the body. Secretion of aldosterone is controlled through the renin-angiotensin system and

hypertrophy of the zona glomerulosa is generally considered to be an adaptative process following stimulation of this system.

 

There were no test item related microscopic findings in the reproductive organs, including the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) and the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries. There were no test item related microscopic findings in the males or females suspected of reduced fertility.

 

A reduction in corpora lutea count was observed in females ingroup 4 during the macroscopic evaluation at necropsy whereas histopathological evaluation of follicles and corpora lutea in the ovaries did not reveal any test item-related effect. Because no significant changes in the number of implantation sites or litter size were observed during the study, the reduction in corpora lutea count was considered not to be adverse. Further, the effect on corpora lutea count may possibly be secondary to the toxic effects in females observed in this group. Remaining reproduction parameters: mating performance, fertility, duration of gestation, post implantation and postnatal loss were not affected by the treatment with the test item at any dose level.

 

With exception for a slight retardation in pup body weights in the presence of adversely reduced food consumption and body weight in females in group 4, no effects on development were observed during the study. The changes in pup body weights were only minor (not statistically significant if compared to the control group), and values in the high-dose group were close to the normal background range in this rat strain. The reduction in pup body weights represents a delayed growth rather than a developmental disturbance. This effect is commonly seen in the presence of maternal toxicity manifested as decreased food consumption associated with reduced body weights and it is considered to be secondary to the maternal toxicity. For these reasons, effects on pup body weights were considered not to be adverse and likely to be a secondary response.

 

Due to the histopathological changes in adrenal glands, no NOEL (No Observed Adverse Effect Level) for general toxicity was established for females whereas for males it was at the dose level of 1000 ppm. The NOAEL (No Observed Adverse Effect Level) was established at 3000 ppm. For reproduction and developmental toxicity, the NOEL was established at the dose level of 3000 ppm whereas the NOAEL was at 10000 ppm, the highest dose level used in the study.