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:
2013-09-18 to 2015-10-02
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
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2013-09-18 to 2015-10-02
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.
Reason / purpose:
read-across source
Reason / purpose:
reference to same study
Remarks:
Sections 7.5.1. 7.8.2.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs associated with dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gain for both sexes, including for females during the gestation and lactation phases of the study, was unaffected by dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption for both sexes, including for females during the gestation and lactation phases of the study, was unaffected by dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.

Food efficiency:
no effects observed
Description (incidence and severity):
Food conversion efficiency for both sexes during the pre-pairing phase of the study and males during the post-pairing phase of the study was unaffected by dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of the water bottles did not indicate any obvious effect on water intake for either sex at 3000, 7500 and 18000/15000 ppm.
Haematological findings:
no effects observed
Description (incidence and severity):
Haematology parameters for both sexes were unaffected by dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
For males at 18000 ppm, higher levels of alanine aminotransferase attained statistical significance when compared with control, however only one individual value exceeded the historical control range and, in the absence of any supporting adverse histopathological findings, this increase was considered to be of little toxicological significance. Additionally for males at this dietary level, higher levels of alkaline phosphatase and urea also attained statistical significance when compared with control; all individual values were within the historical control range and these increases were considered to be of no toxicological significance. Males at 7500 and 18000 ppm showed statistically significant lower potassium levels compared with control but no dosage relationship was apparent. This decrease was considered to reflect high control values, with two individual control values exceeding the historical control range compared to only one value for all the treated males, and was unrelated to dietary exposure to the Test Item.No statistically significant differences from control were apparent for blood chemistry parameters for both sexes receiving 3000 ppm and for females receiving 7500 or 18000/15000 ppm.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioral Assessments: There were no treatment-related changes in the behavioural parameters at 3000, 7500 or 18000/15000 ppm.

Functional Performance Tests: There were no changes in functional performance considered to be related to treatment at 3000, 7500 and 18000/15000 ppm. For females at 7500 and 18000/15000 ppm, superior forelimb grip strength during trial three
attained statistical significance when compared with control but there was no dosage relationship. In the absence of any similar statistically significant differences from control in the previous two trials for forelimb grip strength or for trials of hind limb grip strength, this finding was considered incidental and unrelated to treatment.

Sensory Reactivity Assessments: There were no inter-group differences in sensory reactivity scores apparent during the study.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At 18000/15000 ppm, both sexes showed increased absolute and body weight relative liver weights compared with control with differences attaining statistical significance. This finding was consistent with the minimal centrilobular hepatocellular hypertrophy observed microscopically at this dietary level, although the majority of individual liver values were within the historical control range. Higher absolute and body weight relative liver weights for males at 3000 ppm and at 7500 ppm also attained statistical significance when compared with control; differences showed no consistent dosage relationship and, again, the majority of individual values were within the historical control range. For males at 3000, 7500 and 18000/15000 ppm higher absolute and body weight relative kidney weights attained statistical significance when compared with control, although differences showed no dosage relationship. In the absence of any histopathological correlates, this finding was considered to be of no toxicological significance. For males at 3000 ppm, absolute and relative thyroid weights were higher than control, with differences attaining statistical significance. In the absence of any histopathological change or similar increase at higher dietary levels this finding was considered incidental and unrelated to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for adult animals did not indicate any effect of dietary exposure to the Test Item for either sex at 3000, 7500 and 18000/15000 ppm
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathological changes considered to be associated with exposure to the Test Item were restricted to the liver of both sexes at 18000/15000 ppm, with minimal centrilobular hepatocellular hypertrophy being observed in 5/12 males and 6/12 females at this dietary level. This finding correlated with statistically significantly higher mean absolute and relative liver weight for both sexes but, in the absence of any other degenerative changes in the liver this finding was considered an adaptive change probably resulting from enzyme induction. Although significant statistical differences in mean liver weights were apparent for males at 3000 or 7500 ppm, no corresponding findings were observed in the liver of either sex at these lower
dietary levels. Mild diffuse urothelial hyperplasia was observed for one female at 18000/15000 ppm. While this was considered an unusual change, its occurrence in one animal was considered insufficient to indicate a definite relationship to treatment and, as no similar findings were apparent for any other animal on the study it was concluded that this finding most probably arose spontaneously.All of the other histopathological findings encountered were considered to have occurred
spontaneously or post mortem.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Fertility: Fertility as assessed by pregnancy rate was unaffected by dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.

Gestation Length: The distribution of gestation lengths for treated females did not indicate any effect of dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatmen trelated abnormalities were noted.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating: There were no effects on mating performance at 3000, 7500 or 18000/15000 ppm.

Numbers of corpora lutea and implantations, pre- and post implantation losses, litter size at birth/Day 1, subsequent offspring survival to Day 4 and sex ratio were unaffected by maternal dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Dose descriptor:
NOAEL
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Systemic Toxicity
Dose descriptor:
NOAEL
Effect level:
18 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Systemiic Toxicity
Dose descriptor:
NOEL
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Reproductive Toxicity
Dose descriptor:
NOEL
Effect level:
18 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Reproductive Toxicity
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs apparent for offspring on the study were typical for the age observed and the incidence/distribution of these observations did not indicated any effect of maternal dietary exposure to the Test Item on offspring development at 3000, 7500 or 18000/15000 ppm
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Offspring bodyweight on Day 1 and subsequent body weight gain to Day 4 post partum were unaffected by maternal dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Sexual maturation:
no effects observed
Description (incidence and severity):
Litter size at birth/Day 1, subsequent offspring survival to Day 4 and sex ratio were unaffected by maternal dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings apparent for offspring were typical for the age observed and the low incidence and distribution of these observations did not indicate any effect of maternal dietary exposure to the Test Item on offspring development at 3000, 7500 and 18000/15000 ppm.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Surface righting on Day 1/Day 4 post partum was unaffected by maternal dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Reproductive/Developmental Toxicity
Dose descriptor:
NOEL
Generation:
F1
Effect level:
18 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Reproductive/Developmental Toxicity
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
Based on the results for this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 18000/15000 ppm. There was no obvious effect of treatment on the reproductive parameters within the study therefore the No Observed Effect Level (NOEL) for reproduction was also considered to be 18000/15000 ppm.
Executive summary:

This data is being read across from the source study that tested Resin acids and Rosin acids, esters with ethylene glycol based on category read across that is explained in the category justification document attached in Section 13 of the dossier.

In a key combined repeated dose, reproductive/developmental toxicity study, the test material (Rosin, Ethylene Glycol Ester, CAS # 68512-65-2) was administered by continuous dietary admixture to Wistar Han™:RccHan™:WIST strain rats (12/sex/concentration) for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dietary concentrations of 3000, 7500 and 18000 ppm (equivalent to a mean achieved dosage of 176.3, 457.7 and 1094.1 mg/kg bw/day respectively for males and 215.1, 555.2 and 1272.7 mg/kg bw/day respectively for females during the pre-pairing phase). The dietary concentration given to the high dosage females during gestation and lactation was decreased to 15000 ppm to lessen the expected increase in achieved intake during these phases. A control group of twelve males and twelve females were treated with basal laboratory diet.

 

Clinical signs, body weight gain food consumption and haematology parameters appeared unaffected by exposure to the test material. Higher levels of alanine aminotransferase, alkaline phosphatase and urea were observed for males at 18000 ppm compared to control but individual values were nearly all within the historical control range. These increases are probably associated with adaptive liver changes and were considered not to represent an adverse effect of treatment.

 

Microscopic evaluation of the tissues on the study revealed minimal centrilobular hepatocellular hypertrophy of the liver for males at 18000 ppm and females at 18000/15000 ppm, which was probably the underlying cause for increased liver weight for both sexes at these dietary levels compared to control. The centrilobular hepatocellular hypertrophy was considered to be an adaptive change, probably resulting from enzyme induction resulting from high-dose administration of a xenobiotic, and did not represent an adverse effect of treatment. Increased liver weights were also observed for males receiving 3000 or 7500 ppm and probably reflects a similar, but less significant, adaptive response to exposure.

 

Higher absolute and body weight relative kidney weights compared to control were observed in treated males at all dietary levels but differences showed no dosage relationship and were within the historical range. In the absence of any microscopic kidney changes, these increased weights were considered to be incidental and of no toxicological significance.

 

Based on the results for this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was determined to be 18000/15000 ppm. There was no obvious effect of treatment on the reproductive parameters within the study. Therefore, the No Observed Effect Level (NOEL) for reproduction was determined to be 18000/15000 ppm.

Reason / purpose:
reference to same study
Remarks:
Sections 7.5.1. 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 to have had no impact on the scientific integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
other: Han™:RccHan™:WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd. (Blackthorn, Bicester, Oxon, UK)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 12 wks
- Weight at study initiation: (P) Males: 289 to 343 g; Females:184 to 223 g
- Fasting period before study: Not specified
- Housing: Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): Ground diet (Rodent PMI 5002 (Certified), BCM IPS Limited, London, UK) ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: 2013-10-31 To: 2013-12-20

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtureswere prepared prior to the first treatment and fortnightly thereafter.
- Mixing appropriate amounts with (Type of food): A known amount of the test item was mixed with a small amount of basal laboratory diet until homogeneous in a Robot Coupe Blixer 4 set at a constant speed. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1, in a Hobart H800 mixer.
- Storage temperature of food: The diet was stored in labelled, double plastic bags in labelled, covered plastic bins at room temperature.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- 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:
The stability and uniformity of distribution of the test item in the diet were determined by Harlan Laboratories Ltd. (Shardlow, UK, Analytical Services). Representive samples were taken fron the dietary admixtures and analysed for uniformity of distribution and concentration at Harlan Laboratories Ltd. (Shardlow, UK, Analytical Services).
Duration of treatment / exposure:
up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Frequency of treatment:
continuous dietary admixture
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control (Group 1)
Dose / conc.:
3 000 ppm
Remarks:
Low Concentration (Group 2)
Dose / conc.:
7 500 ppm
Remarks:
Intermediate Concentration (Group 3)
Dose / conc.:
18 000 ppm
Remarks:
High Concentration (Group 4)
The dietary concentration given to the high dosage females during gestation and lactation was decreased to 15000 ppm to lessen the expected increase in achieved intake during these phases.
No. of animals per sex per dose:
12/sex/concentration
Control animals:
yes
Details on study design:
- Dose selection rationale: Dietary levels of 3000, 7500 and 18000 ppm (lowered to 15000 ppm for females during gestation and lactation) were selected, in collaboration with the sponsor, based on available toxicity data including a fourteen day range-finding toxicity study (Harlan Study Number 41302559).
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

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
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

OTHER:

- Functional Observations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

1) Behavioral Assessments: Parameters listed in Table 2. were observed.
2) Functional Performance Tests: Motor Activity and Forelimb/Hindlimb Grip Strength were determined
3) Sensory Reactivity: Parameters listed in Table 2. were observed.

- Hematology and Clinical Biochemistry: Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling. The parameters listed in Tables 3. and 4. were observed.
Oestrous cyclicity (parental animals):
- Pregnancy and Parturition: Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at least twice (early morning and as late as possible during the normal working day) at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

-Litter Data: On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)
vi. All live offspring were assessed for surface righting reflex on Day 1 post partum.
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, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43 or Day 44
- Maternal animals: All surviving adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

GROSS NECROPSY
- All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced for non-pregnant females. The corpora lutea were also counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 5. and 6. were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:
See "any other information on materials an methods incl. tables" for information on statistics.
Reproductive indices:
Mating Performance and Fertility:The following parameters were calculated from the individual data during the mating period of the parental generation:
1) Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
2) Fertility Indices: For each group the following were calculated:

i) Mating Index (%) = (number of animals mated/number of animals paired) x 100
ii) Pregnancy Index (%) = (Number of pregnant females/number of females mated) x 100

2) Gestation and Parturition Data: The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

1) Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
2) Parturition Index: The following was calculated for each group:
i) Parturition Index (%) = (Number of females delivering live offspring/Number of pregnant females) x 100


Offspring viability indices:
Litter Responses: Values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

1) Implantation Losses (%): Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

i. Pre–implantation loss (%) = ((Number of corpora lutea - Number of Implantation Sites)/Number of corpora lutea) x 100
ii) Post–implantation loss (%) = ((Number of implantation sites - Total number of offspring born)/Number of implantation sites) x 100

2) Live Birth and Viability Indices: The following indices were calculated for each litter as follows:

i. Live Birth Index (%) = (Number of offspring alive on Day 1/Number of offspring born) x 100
ii. Viability Index (%) = (Number of offspring alive on Day 4/Number of offsrping alive on Day 1) x 100

3) Sex Ratio (% males): Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:

i. Sex Ratio (% males) = (Number of male offspring/Total number of offspring) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs associated with dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gain for both sexes, including for females during the gestation and lactation phases of the study, was unaffected by dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption for both sexes, including for females during the gestation and lactation phases of the study, was unaffected by dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.

Food efficiency:
no effects observed
Description (incidence and severity):
Food conversion efficiency for both sexes during the pre-pairing phase of the study and males during the post-pairing phase of the study was unaffected by dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of the water bottles did not indicate any obvious effect on water intake for either sex at 3000, 7500 and 18000/15000 ppm.
Haematological findings:
no effects observed
Description (incidence and severity):
Haematology parameters for both sexes were unaffected by dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
For males at 18000 ppm, higher levels of alanine aminotransferase attained statistical significance when compared with control, however only one individual value exceeded the historical control range and, in the absence of any supporting adverse histopathological findings, this increase was considered to be of little toxicological significance. Additionally for males at this dietary level, higher levels of alkaline phosphatase and urea also attained statistical significance when compared with control; all individual values were within the historical control range and these increases were considered to be of no toxicological significance. Males at 7500 and 18000 ppm showed statistically significant lower potassium levels compared with control but no dosage relationship was apparent. This decrease was considered to reflect high control values, with two individual control values exceeding the historical control range compared to only one value for all the treated males, and was unrelated to dietary exposure to the Test Item.No statistically significant differences from control were apparent for blood chemistry parameters for both sexes receiving 3000 ppm and for females receiving 7500 or 18000/15000 ppm.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioral Assessments: There were no treatment-related changes in the behavioural parameters at 3000, 7500 or 18000/15000 ppm.

Functional Performance Tests: There were no changes in functional performance considered to be related to treatment at 3000, 7500 and 18000/15000 ppm. For females at 7500 and 18000/15000 ppm, superior forelimb grip strength during trial three
attained statistical significance when compared with control but there was no dosage relationship. In the absence of any similar statistically significant differences from control in the previous two trials for forelimb grip strength or for trials of hind limb grip strength, this finding was considered incidental and unrelated to treatment.

Sensory Reactivity Assessments: There were no inter-group differences in sensory reactivity scores apparent during the study.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At 18000/15000 ppm, both sexes showed increased absolute and body weight relative liver weights compared with control with differences attaining statistical significance. This finding was consistent with the minimal centrilobular hepatocellular hypertrophy observed microscopically at this dietary level, although the majority of individual liver values were within the historical control range. Higher absolute and body weight relative liver weights for males at 3000 ppm and at 7500 ppm also attained statistical significance when compared with control; differences showed no consistent dosage relationship and, again, the majority of individual values were within the historical control range. For males at 3000, 7500 and 18000/15000 ppm higher absolute and body weight relative kidney weights attained statistical significance when compared with control, although differences showed no dosage relationship. In the absence of any histopathological correlates, this finding was considered to be of no toxicological significance. For males at 3000 ppm, absolute and relative thyroid weights were higher than control, with differences attaining statistical significance. In the absence of any histopathological change or similar increase at higher dietary levels this finding was considered incidental and unrelated to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for adult animals did not indicate any effect of dietary exposure to the Test Item for either sex at 3000, 7500 and 18000/15000 ppm
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathological changes considered to be associated with exposure to the Test Item were restricted to the liver of both sexes at 18000/15000 ppm, with minimal centrilobular hepatocellular hypertrophy being observed in 5/12 males and 6/12 females at this dietary level. This finding correlated with statistically significantly higher mean absolute and relative liver weight for both sexes but, in the absence of any other degenerative changes in the liver this finding was considered an adaptive change probably resulting from enzyme induction. Although significant statistical differences in mean liver weights were apparent for males at 3000 or 7500 ppm, no corresponding findings were observed in the liver of either sex at these lower
dietary levels. Mild diffuse urothelial hyperplasia was observed for one female at 18000/15000 ppm. While this was considered an unusual change, its occurrence in one animal was considered insufficient to indicate a definite relationship to treatment and, as no similar findings were apparent for any other animal on the study it was concluded that this finding most probably arose spontaneously.All of the other histopathological findings encountered were considered to have occurred
spontaneously or post mortem.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Fertility: Fertility as assessed by pregnancy rate was unaffected by dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.

Gestation Length: The distribution of gestation lengths for treated females did not indicate any effect of dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatmen trelated abnormalities were noted.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating: There were no effects on mating performance at 3000, 7500 or 18000/15000 ppm.

Numbers of corpora lutea and implantations, pre- and post implantation losses, litter size at birth/Day 1, subsequent offspring survival to Day 4 and sex ratio were unaffected by maternal dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Systemic Toxicity
Dose descriptor:
NOAEL
Effect level:
18 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Systemiic Toxicity
Dose descriptor:
NOEL
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Reproductive Toxicity
Dose descriptor:
NOEL
Effect level:
18 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Reproductive Toxicity

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs apparent for offspring on the study were typical for the age observed and the incidence/distribution of these observations did not indicated any effect of maternal dietary exposure to the Test Item on offspring development at 3000, 7500 or 18000/15000 ppm
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Offspring bodyweight on Day 1 and subsequent body weight gain to Day 4 post partum were unaffected by maternal dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Sexual maturation:
no effects observed
Description (incidence and severity):
Litter size at birth/Day 1, subsequent offspring survival to Day 4 and sex ratio were unaffected by maternal dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings apparent for offspring were typical for the age observed and the low incidence and distribution of these observations did not indicate any effect of maternal dietary exposure to the Test Item on offspring development at 3000, 7500 and 18000/15000 ppm.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Surface righting on Day 1/Day 4 post partum was unaffected by maternal dietary exposure to the Test Item at 3000, 7500 or 18000/15000 ppm.

Effect levels (F1)

open allclose all
Dose descriptor:
NOEL
Generation:
F1
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Reproductive/Developmental Toxicity
Dose descriptor:
NOEL
Generation:
F1
Effect level:
18 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Reproductive/Developmental Toxicity

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results for this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 18000/15000 ppm. There was no obvious effect of treatment on the reproductive parameters within the study therefore the No Observed Effect Level (NOEL) for reproduction was also considered to be 18000/15000 ppm.
Executive summary:

In a key combined repeated dose, reproductive/developmental toxicity study, the test material (Rosin, Ethylene Glycol Ester, CAS # 68512-65-2) was administered by continuous dietary admixture to Wistar Han™:RccHan™:WIST strain rats (12/sex/concentration) for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dietary concentrations of 3000, 7500 and 18000 ppm (equivalent to a mean achieved dosage of 176.3, 457.7 and 1094.1 mg/kg bw/day respectively for males and 215.1, 555.2 and 1272.7 mg/kg bw/day respectively for females during the pre-pairing phase). The dietary concentration given to the high dosage females during gestation and lactation was decreased to 15000 ppm to lessen the expected increase in achieved intake during these phases. A control group of twelve males and twelve females were treated with basal laboratory diet.

 

Clinical signs, body weight gain food consumption and haematology parameters appeared unaffected by exposure to the test material. Higher levels of alanine aminotransferase, alkaline phosphatase and urea were observed for males at 18000 ppm compared to control but individual values were nearly all within the historical control range. These increases are probably associated with adaptive liver changes and were considered not to represent an adverse effect of treatment.

 

Microscopic evaluation of the tissues on the study revealed minimal centrilobular hepatocellular hypertrophy of the liver for males at 18000 ppm and females at 18000/15000 ppm, which was probably the underlying cause for increased liver weight for both sexes at these dietary levels compared to control. The centrilobular hepatocellular hypertrophy was considered to be an adaptive change, probably resulting from enzyme induction resulting from high-dose administration of a xenobiotic, and did not represent an adverse effect of treatment. Increased liver weights were also observed for males receiving 3000 or 7500 ppm and probably reflects a similar, but less significant, adaptive response to exposure.

 

Higher absolute and body weight relative kidney weights compared to control were observed in treated males at all dietary levels but differences showed no dosage relationship and were within the historical range. In the absence of any microscopic kidney changes, these increased weights were considered to be incidental and of no toxicological significance.

 

Based on the results for this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was determined to be 18000/15000 ppm. There was no obvious effect of treatment on the reproductive parameters within the study. Therefore, the No Observed Effect Level (NOEL) for reproduction was determined to be 18000/15000 ppm.