Resin acids and Rosin acids, esters with glycerol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-27 to 2017-01-30

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 from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2015-01-27 to 2017-01-30

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

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were apparent during the study at 3000, 7500 or 18000 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):

There was no effect of treatment on body weight gain for either sex at 3000, 7500 or 18000 ppm. Overall body weight gain for males at 7500 ppm was superior to control but this, and occasional statistically significant differences in body weightgain for treated males, 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 effect of treatment on food consumption for either sex at 3000, 7500 or 18000 ppm.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no effect of treatment on food conversion efficiency for either sex at 3000, 7500 or 18000 ppm.

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

no effects observed

Description (incidence and severity):

There was no observed effect of treatment on water consumption for either sex at dosages of 3000, 7500 or 18000 ppm.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

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

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects on hematology parameters were observed in either sex at 3000, 7500 or 18000 ppm.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on blood chemistry parameters were observed in either sex at 3000, 7500 or 18000 ppm.

For males at 18000 ppm, lower albumin and total protein and higher cholesterol and bilirubin attained statistical significance (p<0.05) when compared with control. A statistically significant (p<0.05) reduction in albumin was also evident in males treated with 7500 ppm. For females at 18000 ppm, higher bilirubin also attained statistical significance. In the absence of any histopathological correlates, the intergroup differences were considered to be incidental and unrelated to treatment.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioral Assessments
Weekly assessment of the animals in a standard arena did not reveal any effects of treatment at 3000, 7500 or 18000 ppm.

Functional Performance Tests
Assessment of functional performance using grip strength and motor activity did not indicate any effects of treatment for either at 3000, 7500 or 18000 ppm.

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

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was considered to be no adverse effect oftreatment on the organ weights measured for either sex at 3000, 7500 or 18000 ppm.

In animals of either sex treated with 18000 ppm and males treated with 7500 ppm, liver weights (absolute and relative to body weight) were statistically significantly higher (p<0.05 for females treated with 18000 ppm and for males treated with 7500 ppm, p<0.01 for males treated with 18000 ppm) than control. In the absence of any changes indicative of a pathological effect, this increase in organ weight was considered to be non-adverse and probably adaptive in nature.

No changes were observed in animals of either sex treated with 3000 ppm.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Neither the type, incidence nor distribution of findings observed at terminal necropsy indicated any obvious effect of treatment 3000, 7500 or 18000 ppm.

Histopathological findings: non-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 18000 ppm indicated any effect of treatment.

Dose descriptor:

NOEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on nonadverse absolute and relative liver weight changes in males at 7500 ppm and both sexes at 15000 ppm.

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 results of this ninety day study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of Rosin, ethylene glycol ester CAS 68512-65-2 was considered to be 18000 ppm (equivalent to 1202.5 mg/kg bw/day for males and 1377.8 mg/kg bw/day for
females). The No Observed Effect Level (NOEL) was considered to be 3000 ppm based on nonadverse absolute and relative liver weight changes in males at 7500 ppm and both sexes at 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 repeated dose oral toxicity study, the test material (Rosin, esters with ethylene glycol; CAS# 68512-65-2) was administered by continuous dietary admixture to three groups, each composed of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dietary concentrations of 3000, 7500 and 18000 ppm (equivalent to a mean achieved dosage of 205.7, 506.6 and 1202.5 mg/kg bw/day for males and 243.2, 594.4 and 1377.8 mg/kg bw/day for females). A control group of ten males and ten females were fed basal laboratory diet only.

 

Clinical signs, functional observations, bodyweight change, dietary intake and water consumption were monitored during the study. Hematology 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 Week 12 of the study. All animals were subjected to gross necropsy examination and a comprehensive histopathological evaluation of tissues from high dose and control animals was performed.

 

The continuous oral (dietary) administration of the test material in the diet for ninety consecutive days was well tolerated and was not associated with any obvious signs of systemic toxicity for animals of either sex. There were no effects of dietary exposure indicated by body weight performance, food and water consumption, food conversion efficiency, functional observations or ophthalmic examinations. Haematological investigations did not reveal any adverse effect of treatment; however, blood chemical evaluations did reveal a number of statistically significant intergroup differences. Males fed diet containing 18000 ppm showed lower albumin and total protein and higher cholesterol and bilirubin levels. The effect on albumin was also evident in males fed diet containing 7500 ppm. Higher bilirubin levels were also evident in females fed diet containing 18000 ppm. In animals of either sex fed diet containing 18000 ppm and males fed diet containing 7500 ppm, liver weights both absolute and relative to terminal body weight were higher than controls. These intergroup differences in blood chemistry and organ weights may be a consequence of enhanced hepatic metabolism and turnover as a by-product of xenobiotic metabolism. However, due to the lack of pathological changes in the liver, these intergroup differences may represent altered metabolic function but these occurred in the absence of adverse gross or microscopic hepatic effects after ninety consecutive days of dietary exposure.

 

Based on the results of this ninety-day study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 18000 ppm (equivalent to 1202.5 mg/kg bw/day for males and 1377.8 mg/kg bw/day for females). The No Observed Effect Level (NOEL) was considered to be 3000 ppm based on non adverse absolute and relative liver weight changes in males at 7500 ppm and both sexes at 15000 ppm.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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 scientific purpose of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Rosin ethylene glycol ester (CAS RN 68512-65-2)

IUPAC Name:

Rosin ethylene glycol ester (CAS RN 68512-65-2)

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

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 in safety evaluation studies and is acceptable to appropriate 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: 183 to 233 g; Females: 150 to 184 g
- Fasting period before study: not specified
- Housing: 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, Essex, UK) was used ad libitum.
- Water (e.g. ad libitum): Mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage.
- Acclimation period: Nine or ten days for males and females, respectively.

DETAILS OF FOOD AND WATER QUALITY: Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). 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): At 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: 2015-01-30 To: 2015-05-01

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were prepared approximately every fourteen days
- 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 thirty minutes at a constant speed, setting 1 in a Hobart QE200 mixer.
- Storage temperature of food: Stored at room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity of the dietary admixtures was previously performed under Envigo Research Limited, Study Number 41302558; Oral (Dietary) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening in the Rat (OECD 422). The stability of the dietary admixtures was determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the dietary admixtures to be stable for at least 27 days at room temperature. Dietary admixtures were therefore prepared approximately every fourteen days and stored at room temperature. Representative samples were taken of dietary admixtures on three occasions during the study and analyzed for concentration of Rosin, ethylene glycol ester CAS 68512-65-2 at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 93 % to 125 % of the nominal concentration confirming the suitability and accuracy of the formulation procedure.

Duration of treatment / exposure:

The test item was administered continuously, for ninety consecutive days, by dietary admixture.

Frequency of treatment:

Administered continuously by dietary admixture

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

3 000 ppm

Remarks:

Low Concentration
(equivalent to 205.7 and 243.2 mg/Kg bw/day for males and females, respectively)

Doses / concentrations 3

Dose / conc.:

7 500 ppm

Remarks:

Intermediate Concentration
(equivalent to 506.6 and 594.4 mg/Kg bw/day for males and females, respectively)

Doses / concentrations 4

Dose / conc.:

18 000 ppm

Remarks:

High Concentration
(equivalent to 1202.5 and 1377.8 mg/Kg bw/day for males and females, respectively)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary concentrations were chosen based onthe results of previous toxicity work (Envigo Research Limited, Study Number 41302558; Oral (Dietary) Combined Repeat Dose Toxicity Study with Reproduction/Development Toxicity Screening Test in the Rat (OECD 422)). 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 believed to be of value in evaluating the potential hazards of the test item to man.
- 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 for cross contamination of the treated diets. 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: Daily

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

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: 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: 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 detailed individual clinical signs outside the home cage in a standard arena. These arena observations commenced between 10.00 and 14.30. During Week 12, these observations were combined with functional performance tests and assessment of sensory reactivity to different stimuli to represent a full Functional Observation Battery.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Parameters checked in table [No.4] were examined.

IMMUNOLOGY: No

Sacrifice and pathology:

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

GROSS PATHOLOGY: Yes (see table 5)

HISTOPATHOLOGY: Yes (see table 6)

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, 7500 or 18000 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):

There was no effect of treatment on body weight gain for either sex at 3000, 7500 or 18000 ppm. Overall body weight gain for males at 7500 ppm was superior to control but this, and occasional statistically significant differences in body weightgain for treated males, 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 effect of treatment on food consumption for either sex at 3000, 7500 or 18000 ppm.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no effect of treatment on food conversion efficiency for either sex at 3000, 7500 or 18000 ppm.

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

no effects observed

Description (incidence and severity):

There was no observed effect of treatment on water consumption for either sex at dosages of 3000, 7500 or 18000 ppm.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

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

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects on hematology parameters were observed in either sex at 3000, 7500 or 18000 ppm.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on blood chemistry parameters were observed in either sex at 3000, 7500 or 18000 ppm.

For males at 18000 ppm, lower albumin and total protein and higher cholesterol and bilirubin attained statistical significance (p<0.05) when compared with control. A statistically significant (p<0.05) reduction in albumin was also evident in males treated with 7500 ppm. For females at 18000 ppm, higher bilirubin also attained statistical significance. In the absence of any histopathological correlates, the intergroup differences were considered to be incidental and unrelated to treatment.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioral Assessments
Weekly assessment of the animals in a standard arena did not reveal any effects of treatment at 3000, 7500 or 18000 ppm.

Functional Performance Tests
Assessment of functional performance using grip strength and motor activity did not indicate any effects of treatment for either at 3000, 7500 or 18000 ppm.

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

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was considered to be no adverse effect oftreatment on the organ weights measured for either sex at 3000, 7500 or 18000 ppm.

In animals of either sex treated with 18000 ppm and males treated with 7500 ppm, liver weights (absolute and relative to body weight) were statistically significantly higher (p<0.05 for females treated with 18000 ppm and for males treated with 7500 ppm, p<0.01 for males treated with 18000 ppm) than control. In the absence of any changes indicative of a pathological effect, this increase in organ weight was considered to be non-adverse and probably adaptive in nature.

No changes were observed in animals of either sex treated with 3000 ppm.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Neither the type, incidence nor distribution of findings observed at terminal necropsy indicated any obvious effect of treatment 3000, 7500 or 18000 ppm.

Histopathological findings: non-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 18000 ppm indicated any effect of treatment.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on nonadverse absolute and relative liver weight changes in males at 7500 ppm and both sexes at 15000 ppm.

2

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 results of this ninety day study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of Rosin, ethylene glycol ester CAS 68512-65-2 was considered to be 18000 ppm (equivalent to 1202.5 mg/kg bw/day for males and 1377.8 mg/kg bw/day for
females). The No Observed Effect Level (NOEL) was considered to be 3000 ppm based on nonadverse absolute and relative liver weight changes in males at 7500 ppm and both sexes at 15000 ppm.

Executive summary:

In a key repeated dose oral toxicity study, the test material (Rosin, esters with ethylene glycol; CAS# 68512-65-2) was administered by continuous dietary admixture to three groups, each composed of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dietary concentrations of 3000, 7500 and 18000 ppm (equivalent to a mean achieved dosage of 205.7, 506.6 and 1202.5 mg/kg bw/day for males and 243.2, 594.4 and 1377.8 mg/kg bw/day for females). A control group of ten males and ten females were fed basal laboratory diet only.

 

Clinical signs, functional observations, bodyweight change, dietary intake and water consumption were monitored during the study. Hematology 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 Week 12 of the study. All animals were subjected to gross necropsy examination and a comprehensive histopathological evaluation of tissues from high dose and control animals was performed.

 

The continuous oral (dietary) administration of the test material in the diet for ninety consecutive days was well tolerated and was not associated with any obvious signs of systemic toxicity for animals of either sex. There were no effects of dietary exposure indicated by body weight performance, food and water consumption, food conversion efficiency, functional observations or ophthalmic examinations. Haematological investigations did not reveal any adverse effect of treatment; however, blood chemical evaluations did reveal a number of statistically significant intergroup differences. Males fed diet containing 18000 ppm showed lower albumin and total protein and higher cholesterol and bilirubin levels. The effect on albumin was also evident in males fed diet containing 7500 ppm. Higher bilirubin levels were also evident in females fed diet containing 18000 ppm. In animals of either sex fed diet containing 18000 ppm and males fed diet containing 7500 ppm, liver weights both absolute and relative to terminal body weight were higher than controls. These intergroup differences in blood chemistry and organ weights may be a consequence of enhanced hepatic metabolism and turnover as a by-product of xenobiotic metabolism. However, due to the lack of pathological changes in the liver, these intergroup differences may represent altered metabolic function but these occurred in the absence of adverse gross or microscopic hepatic effects after ninety consecutive days of dietary exposure.

 

Based on the results of this ninety-day study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 18000 ppm (equivalent to 1202.5 mg/kg bw/day for males and 1377.8 mg/kg bw/day for females). The No Observed Effect Level (NOEL) was considered to be 3000 ppm based on non adverse absolute and relative liver weight changes in males at 7500 ppm and both sexes at 15000 ppm.