Rosin, maleated, esterified with sorbitol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-01 to 2015-09-03

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-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2015-04-01 to 2015-09-03

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 for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

deviations had no adverse impact on the results of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Hydrocarbons Resins and Rosin Resins REACH Consortium (Brussels, Belgium); Batch no: None - continuous manufacture ex WFE, sample taken 3 November 2014
- Expiration date of the lot/batch: Not supplied. The test item does not deteriorate if stored in the freezer or fridge
- Purity test date: 2014-11-06

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored frozen at ~ -20°C, in the dark, under Nitrogen

FORM AS APPLIED IN THE TEST (if different from that of starting material)

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used for the evaluation of chemical hazards and is considered acceptable by regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: Males - 195 to 237g; Females - 147 to 181g
- Fasting period before study: Not specified
- Housing: The animals were housed in groups of up to four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): Ground diet (Rat and Mouse SQC Ground Diet No. 1, Special Diet Services, DietexInternational Limited, Witham, Essess, UK) ad libitum.
- Water (e.g. ad libitum): Mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage.
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): t least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: 2015-04-24 To: 2015-07-24

Route of administration:

oral: feed

Details on route of administration:

The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are considered to be relevant for the evaluation of the toxicological properties of the test item.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were initially prepared weekly for the first six weeks and then fortnightly thereafter and stored at room temperature
- Mixing appropriate amounts with (Type of food): A known amount of test item was mixed with a small amount of basal laboratory diet in a Robot Coupe Blixer 4 mixer until homogeneous. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further sixty minutes at a constant speed, setting 1 in a Hobart QE200 mixer.
- Storage temperature of food: Room Temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the dietary admixtures was determined by Harlan Laboratories Ltd. (Shardlow, UK, Analytical Services). Representative samples of dietary admixtures were taken and analyzed for concentration of Resin acids and Rosin acids, fumarated, esters with pentaerythitol CAS 94581-15-4 at Harlan Laboratories Ltd. (Shardlow, UK, Analytical Services).

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously in the diet

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

300 ppm

Remarks:

Low concentration

Dose / conc.:

6 000 ppm

Remarks:

Intermediate concentration

Dose / conc.:

18 000 ppm

Remarks:

High concentration
Dietary concentration was 12000 ppm for the first two weeks, 15000 ppm for the next four weeks and then 18000 ppm for the remainder of the study.

No. of animals per sex per dose:

10/sex/concemtration

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dietary concentrations were chosen in collaboration with the Sponsor and based on available toxicity data.
- 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 cages were distributed in dose group columns within the holding rack to minimize the potential of cross contamination of the treated diet. The animals were uniquely identified within the study by an ear punching system routinely used in
these laboratories.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overtsigns of toxicity, ill-health or behavioral change daily from the start of treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior the start of treatment) and at weekly intervals thereafter. 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
Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and before termination of treatment (during Week 12)
- Dose groups that were examined: all control and high dose animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of the study (Day 90)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of the study (Day 90)
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters checked in table [No.3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 12, together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity
- Parameters checked in table [No. 4] were examined.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 5)

HISTOPATHOLOGY: Yes (see table 6)

On completion of the dosing period all animals werekilled by intravenous overdose of a suitable barbiturate agent followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the MannWhitney U test (non-parametric). Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were apparent during the study at 3000, 6000 or 18000 ppm.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Dietary exposure to 3000, 6000 or 18000 ppm produced no adverse effects on body weight gain for either sex. Overall body weight gain for treated males was slightly higher than the control group and occasional statistically significant reductions in body weight gain for treated females relative to control were considered to reflect normal biological variation and were unrelated to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no adverse effect of dietary exposure to 3000, 6000 or 18000 ppm of the test item on food consumption for either sex.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no adverse effect of dietary exposure to 3000, 6000 or 18000 ppm of the test item on food conversion efficiency for either sex.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

There was no observed effect of dietary exposure to 3000, 6000 or 18000 ppm of the test item on water consumption for either sex.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmic examination of the eyes from rats receiving diets containing 18000 ppm did not indicate any effect of treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected in the hematological parameters examined.

Males from all dietary exposure groups showed a statistically significant reduction (p<0.05) in erythrocytes and a statistically significant increase (p<0.05) in mean corpuscular volume. Females from all treatment groups showed statistically significant reductions in mean corpuscular hemoglobin (p<0.05) and mean corpuscular volume (p<0.05-0.01). The majority of individual values for either sex in these parameters were within the historical control range and in the absence of any histopathological correlates or true dose related responses, the intergroup differences were considered to be incidental and unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined.

Females in the 18000 ppm group showed a statistically significant increase (p<0.05) in chloride concentration. Females in the 3000 ppm group showed a statistically significant reduction (p<0.05) in alanine aminotransferase. All of the individual values were within the historical control ranges and in the absence of a true dose related response or any associated histopathological correlates, the intergroup differences were not considered to be toxicologically significant.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioural Assessments : Weekly assessment of the animals in a standard arena did not reveal any obvious adverse effects of dietary exposure to 3000, 6000 or 18000 ppm of the test item.

Functional Performance Tests: There were no toxicologically significant effects detected in functional performance. Males treated with 6000 ppm showed a statistically significant increase in overall activity (p<0.01). In the absence of a true dose related response or any supporting clinical observations or pathological change to suggest a neurotoxiceffect, the intergroup difference was considered to be of no toxicological significance.

Sensory Reactivity Assessments: There were no differences observed in the scoresfor sensory reactivity for either sex during the study.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was considered to be no effect of dietary exposure to 3000, 6000 or 18000 ppm on the organ weights in either sex.

Absolute and relative liver weights were statistically significantly lower than control in males from all treatment groups. The majority of the individual values were within the historical control range and in the absence of any histopathological correlates ora true dose-related response, these intergroup differences were considered of no toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Neither the type, incidence nor distribution of findings observed at terminal necropsy indicated any obvious effect of dietary exposure to 3000, 6000 or 18000 ppm of the test item.

Incidences of reddened lungs were noted in a number of control and treated females and in one male in the 3000 ppm group at necropsy. These were considered to be incidental findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Neither the type, incidence nor distribution of findings observed during microscopic examination of the tissues from animals that received diets containing 18000 ppm of the test item indicated any effect of treatment.

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

18 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic Toxicity

Critical effects observed:

no

Conclusions:

Based on the lack of adverse treatment-related effects observed through the study period, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity for both sexes was determined to be 18000 ppm (equivalent to a mean achieved dosage of 1090.0 mg/kg bw/day for males and 1298.9 mg/kg bw/day for females).

Executive summary:

In a key oral sub-chronic toxicity study, the test material (Resin acids and Rosin acids,fumarated, esters with pentaerythitol; CAS# 94581-15-4) was administered by continuous dietary exposure to Wistar Han™:RccHan™:WIST strain rats (10/sex/concentration), for ninety consecutive days, at dietary concentrations of 3000 and 6000 ppm for the low and intermediate concentration groups (equivalent to a mean achieved dosage of 210.0 and 414.1 mg/kg bw/day for males and 262.4 and 511.2 mg/kg bw/day for females). For the high concentration group, rats were initially fed diet containing 12000 ppm for two weeks followed by 15000 ppm for four weeks and subsequently 18000 ppm for the remainder of the study (equivalent to a mean achieved dosage of 1090.0 mg/kg bw/day for males and 1298.9 mg/kg bw/day for females). A control group of ten males and ten females were treated with basal laboratory diet.

 

Clinical signs, functional observations, bodyweight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals before the start of treatment and during study week 12. All animals were subjected to gross necropsy examination and a comprehensive histopathological evaluation of tissues from high concentration and control animals was also performed.

 

The continuous oral (dietary) administration of the test material for ninety consecutive days, did not result in any toxicologically significant effects. Therefore, based on the lack of adverse treatment-related effects observed through the study period, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity for both sexes was determined to be 18000 ppm (equivalent to a mean achieved dosage of 1090.0 mg/kg bw/day for males and 1298.9 mg/kg bw/day for females).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

deviations had no adverse impact on the results of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Resin acids and Rosin acids, fumarated, esters with pentaerythritol (CAS RN 94581-15-4)

IUPAC Name:

Resin acids and Rosin acids, fumarated, esters with pentaerythritol (CAS RN 94581-15-4)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Hydrocarbons Resins and Rosin Resins REACH Consortium (Brussels, Belgium); Batch no: None - continuous manufacture ex WFE, sample taken 3 November 2014
- Expiration date of the lot/batch: Not supplied. The test item does not deteriorate if stored in the freezer or fridge
- Purity test date: 2014-11-06

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored frozen at ~ -20°C, in the dark, under Nitrogen

FORM AS APPLIED IN THE TEST (if different from that of starting material)

Test animals

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used for the evaluation of chemical hazards and is considered acceptable by regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: Males - 195 to 237g; Females - 147 to 181g
- Fasting period before study: Not specified
- Housing: The animals were housed in groups of up to four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): Ground diet (Rat and Mouse SQC Ground Diet No. 1, Special Diet Services, DietexInternational Limited, Witham, Essess, UK) ad libitum.
- Water (e.g. ad libitum): Mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage.
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): t least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: 2015-04-24 To: 2015-07-24

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are considered to be relevant for the evaluation of the toxicological properties of the test item.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were initially prepared weekly for the first six weeks and then fortnightly thereafter and stored at room temperature
- Mixing appropriate amounts with (Type of food): A known amount of test item was mixed with a small amount of basal laboratory diet in a Robot Coupe Blixer 4 mixer until homogeneous. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further sixty minutes at a constant speed, setting 1 in a Hobart QE200 mixer.
- Storage temperature of food: Room Temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the dietary admixtures was determined by Harlan Laboratories Ltd. (Shardlow, UK, Analytical Services). Representative samples of dietary admixtures were taken and analyzed for concentration of Resin acids and Rosin acids, fumarated, esters with pentaerythitol CAS 94581-15-4 at Harlan Laboratories Ltd. (Shardlow, UK, Analytical Services).

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously in the diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

300 ppm

Remarks:

Low concentration

Doses / concentrations 3

Dose / conc.:

6 000 ppm

Remarks:

Intermediate concentration

Doses / concentrations 4

Dose / conc.:

18 000 ppm

Remarks:

High concentration
Dietary concentration was 12000 ppm for the first two weeks, 15000 ppm for the next four weeks and then 18000 ppm for the remainder of the study.

No. of animals per sex per dose:

10/sex/concemtration

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dietary concentrations were chosen in collaboration with the Sponsor and based on available toxicity data.
- 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 cages were distributed in dose group columns within the holding rack to minimize the potential of cross contamination of the treated diet. The animals were uniquely identified within the study by an ear punching system routinely used in
these laboratories.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overtsigns of toxicity, ill-health or behavioral change daily from the start of treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior the start of treatment) and at weekly intervals thereafter. 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
Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and before termination of treatment (during Week 12)
- Dose groups that were examined: all control and high dose animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of the study (Day 90)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of the study (Day 90)
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters checked in table [No.3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 12, together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity
- Parameters checked in table [No. 4] were examined.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 5)

HISTOPATHOLOGY: Yes (see table 6)

On completion of the dosing period all animals werekilled by intravenous overdose of a suitable barbiturate agent followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the MannWhitney U test (non-parametric). Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were apparent during the study at 3000, 6000 or 18000 ppm.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Dietary exposure to 3000, 6000 or 18000 ppm produced no adverse effects on body weight gain for either sex. Overall body weight gain for treated males was slightly higher than the control group and occasional statistically significant reductions in body weight gain for treated females relative to control were considered to reflect normal biological variation and were unrelated to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no adverse effect of dietary exposure to 3000, 6000 or 18000 ppm of the test item on food consumption for either sex.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no adverse effect of dietary exposure to 3000, 6000 or 18000 ppm of the test item on food conversion efficiency for either sex.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

There was no observed effect of dietary exposure to 3000, 6000 or 18000 ppm of the test item on water consumption for either sex.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmic examination of the eyes from rats receiving diets containing 18000 ppm did not indicate any effect of treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected in the hematological parameters examined.

Males from all dietary exposure groups showed a statistically significant reduction (p<0.05) in erythrocytes and a statistically significant increase (p<0.05) in mean corpuscular volume. Females from all treatment groups showed statistically significant reductions in mean corpuscular hemoglobin (p<0.05) and mean corpuscular volume (p<0.05-0.01). The majority of individual values for either sex in these parameters were within the historical control range and in the absence of any histopathological correlates or true dose related responses, the intergroup differences were considered to be incidental and unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined.

Females in the 18000 ppm group showed a statistically significant increase (p<0.05) in chloride concentration. Females in the 3000 ppm group showed a statistically significant reduction (p<0.05) in alanine aminotransferase. All of the individual values were within the historical control ranges and in the absence of a true dose related response or any associated histopathological correlates, the intergroup differences were not considered to be toxicologically significant.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioural Assessments : Weekly assessment of the animals in a standard arena did not reveal any obvious adverse effects of dietary exposure to 3000, 6000 or 18000 ppm of the test item.

Functional Performance Tests: There were no toxicologically significant effects detected in functional performance. Males treated with 6000 ppm showed a statistically significant increase in overall activity (p<0.01). In the absence of a true dose related response or any supporting clinical observations or pathological change to suggest a neurotoxiceffect, the intergroup difference was considered to be of no toxicological significance.

Sensory Reactivity Assessments: There were no differences observed in the scoresfor sensory reactivity for either sex during the study.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was considered to be no effect of dietary exposure to 3000, 6000 or 18000 ppm on the organ weights in either sex.

Absolute and relative liver weights were statistically significantly lower than control in males from all treatment groups. The majority of the individual values were within the historical control range and in the absence of any histopathological correlates ora true dose-related response, these intergroup differences were considered of no toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Neither the type, incidence nor distribution of findings observed at terminal necropsy indicated any obvious effect of dietary exposure to 3000, 6000 or 18000 ppm of the test item.

Incidences of reddened lungs were noted in a number of control and treated females and in one male in the 3000 ppm group at necropsy. These were considered to be incidental findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Neither the type, incidence nor distribution of findings observed during microscopic examination of the tissues from animals that received diets containing 18000 ppm of the test item indicated any effect of treatment.

Other effects:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

18 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic Toxicity

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the lack of adverse treatment-related effects observed through the study period, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity for both sexes was determined to be 18000 ppm (equivalent to a mean achieved dosage of 1090.0 mg/kg bw/day for males and 1298.9 mg/kg bw/day for females).

Executive summary:

In a key oral sub-chronic toxicity study, the test material (Resin acids and Rosin acids,fumarated, esters with pentaerythitol; CAS# 94581-15-4) was administered by continuous dietary exposure to Wistar Han™:RccHan™:WIST strain rats (10/sex/concentration), for ninety consecutive days, at dietary concentrations of 3000 and 6000 ppm for the low and intermediate concentration groups (equivalent to a mean achieved dosage of 210.0 and 414.1 mg/kg bw/day for males and 262.4 and 511.2 mg/kg bw/day for females). For the high concentration group, rats were initially fed diet containing 12000 ppm for two weeks followed by 15000 ppm for four weeks and subsequently 18000 ppm for the remainder of the study (equivalent to a mean achieved dosage of 1090.0 mg/kg bw/day for males and 1298.9 mg/kg bw/day for females). A control group of ten males and ten females were treated with basal laboratory diet.

 

Clinical signs, functional observations, bodyweight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals before the start of treatment and during study week 12. All animals were subjected to gross necropsy examination and a comprehensive histopathological evaluation of tissues from high concentration and control animals was also performed.

 

The continuous oral (dietary) administration of the test material for ninety consecutive days, did not result in any toxicologically significant effects. Therefore, based on the lack of adverse treatment-related effects observed through the study period, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity for both sexes was determined to be 18000 ppm (equivalent to a mean achieved dosage of 1090.0 mg/kg bw/day for males and 1298.9 mg/kg bw/day for females).