Fatty acids, tall-oil, reaction products with boric acid (H3BO3) and diethanolamine

Endpoint summary

Description of key information

Oral route

In a key OECD 408 Guideline (90-day repeat oral dose) study in rats treatment-related changes were observed in all animals at the 1000 mg/kg bw/day dose level. Effects noted in females at the 250 mg/kg bw/day dose level were considered not to be adverse therefore the NOAEL is considered to be 250 mg/kg bw/day. The NOEL for both sexes is 50 mg/kg bw/day.

Dermal route

In a key 28- day repeat dose dermal toxicity study performed according to OECD TG 410, no mortalities and no evidence of systemic toxicity was seen in any of the groups during the study. Evidence of mild dermal irritation (erythema after the first week of dosing and/or desquamation during the third and fourth week) was seen in two females at 100 mg/kg bw/day, one male and most females at 300 mg/kg bw/day and most animals at 1000 mg/kg bw/day. No severe dermal irritation or evidence of deep tissue damage was apparent. The NOAEL for systemic toxicity was determined to be 1000 mg/kg bw/day. The LOAEL for local effects was 100 mg/kg bw/day based on mild skin irritation observed in 1/5 females.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 March 1998 to 15 December 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Occasionally minor deviations from standard conditions required for room temperature/humidity occurred, but were considered not to have affected the purpose or integrity

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: ca. six to seven weeks
- Weight at study initiation: Males: 168 - 224 g, females: 151 - 192 g
- Fasting period before study: no
- Housing: groups of four by sex in polypropylene gridfloor cages suspended over trays lined with absorbent paper.
- Diet (e.g. ad libitum): Pelleted diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least 15 per hour.
- Photoperiod (hrs dark / hrs light): 12h light/12h dark

IN-LIFE DATES: From: 1998-03-05 To: 1998-12-15

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability of the test material formulations was determined by Safepharm Analytical Laboratory. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4C in the dark.
Samples were taken of each test material formulation and were analysed for concentration of the test substance at Safepharm Analytical Laboratory. The results indicate that the prepared formulations were within 10% of the nominal concentration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Unknown solubility in water
- Concentration in vehicle: 0, 25, 125, 500 mg/mL
- Amount of vehicle (if gavage): 2 mL
- Lot/batch no. (if required): N o data available
- Purity: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC using an external standard.

Samples and standards prepared in methanol to give a concentration of 0.5 mg/mL

HPLC system: Hewlett Packard 1050
Column: Hypersil C18 (150 x 4.6 id)
Mobile phase: Eluent A - water and Eluent B - methanol
At time 0 - 10% B; 14 mins - 100% B and 26 mins - 100% B
Flowrate: 1.5 mL/min
UV detector wavelength: 240 nm
Injection volume: 25 µL
Retention time: 14 to 15 mins

Stability determination: analysed initally and after 14 days in the dark at +4°C.
Stability: 92-96% of initial concentration after 14 days

Formulation concentrations: sampled and analysed within three days of preparation
Formulation: 93 - 109% of nominal

Duration of treatment / exposure:

90-days, consecutive

Frequency of treatment:

One dose per day

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of the range-finding study (0, 150, 500 and 1000 mg/kg/day; 2 ml/kg/day in Arachis
oil BP). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material, and the results of the study are believed to be of value in predicting the likely toxicity of the test material to man. The test material was administered daily, for fourteen consecutive days, by gavage.

- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified bodyweight randomisation procedure and the group mean bodyweights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised.
- Rationale for selecting satellite groups: NA.
- Post-exposure recovery period in satellite groups: no data available.
- Section schedule rationale (if not random): Random

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed immediately before dosing and one hour after dosing at weekends and on public holidays. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends and on public holidays). All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed immediately before dosing and one hour after dosing at weekends and on public holidays. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends and on public holidays). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and at weekly intervals thereafter. Bodyweights 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.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No.

FOOD EFFICIENCY: No data

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex following pupil dilation with 0.5% Tropicamide solution.
- Dose groups that were examined: all control and high dose animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters were examined:
Haemoglobin (Hb), Erythrocyte count (RBC); Haematocrit (Hct); Erythrocyte indices mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC); Total leucocyte count (WBC); Differential leucocyte count: neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas); Platelet count (PLT); Prothrombin time (CT) was assessed by ’Hepato Quick’ and Activated partial thromboplastin time (APTT) was assessed by ’Preci Clot’ using samples collected into sodium citrate solution (0.11 mol/l).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91.
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters were examined: Urea, Inorganic phosphorus (P), GIucose, Total protein (Tot.Prot.) AIbumin, Alanine aminotransferase (ALAT), Albumin/Globulin (NG) ratio (by calculation), Aspartate ami notransferase (ASAT), Alkaline phosphatase (AP), Sodium (Na+), Potassi um (K +), Chloride (CI-), Calcium (Ca+ +), Creatinine (Creat), Total cholesterol (Chol), Total bilirubin (Bili)

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter.
- Dose groups that were examined: all animals were observed for signs of functional/ behavioural toxicity.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the dosing period all animals were killed by intravenous overdose of
sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin:
Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain, Caecum, Colon, Duodenum, Epididymides, Eyes, Gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Mammary glands, Muscle (skeletal), Oesophagus, Seminal vesicles, Skin (hind limb), Spinal cord (cervical, mid-thoracic and lumbar), , Spleen, Stomach, Testes (with epididymides), Thymus, Thyroid/parathyroid, Tougue, Trachea, Urinary bladder, Uterus.
All tissues from control and 1000 mg/kg/d dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesion was also processed.
Since there were indications of treatment-related liver, spleen, thyroid, lung and bone marrow changes, examination was subsequently extended to include similarly prepared sections from all animals in the other treatment groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerization system for tabulation and report production.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight) and weekly bodyweight gain data were assessed for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney "U" Test.
Following statistical analysis of MCHC among control and test animals, statistical significance was assigned to females from the 50 mg/kg/day treatment group in comparison with controls. The difference in mean and standard deviation between these two groups was negligible and the results of this analysis were rejected for contravening the function of Dunnett's pairwise comparison test. A single t-test was performed to compare the control female MCHC values with those of each treatment group in turn and the results of this test were reported accordingly.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.
Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes.
Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
The clinically observable signs detected throughout the study were considered to be without toxicological importance.

Mortality:
There were no deaths during the study.

BODY WEIGHT AND WEIGHT GAIN
A statistically significant reduction in bodyweight gain was detected for 1000 mg/kg/day males during Weeks 2 and 10 when compared with that of controls. Bodyweight development in these animals was unaffected throughout the rest of the study period.
No adverse effect on bodyweight gain was detected for 1000 mg/kg/day females or for animals of either sex treated with 250 or 50 mg/kg/day.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
A slight reduction in dietary intake was detected for 1000 mg/kg/day males over
the first three weeks of treatment. Food efficiency was also slightly reduced during Week 10, corresponding to the bodyweight reduction seen at this time.
No adverse effect on food consumption or efficiency was detected for 1000 mg/kg/day females or for animals of either sex treated with 250 or 50 mg/kg/day throughout the study period.

WATER CONSUMPTION
Daily visual inspection of water bottles revealed no intergroup differences.

OPHTHALMOSCOPIC EXAMINATION:
There were no treatment-related ocular effects.
The incidental abnormalities recorded for two control males were consistent with occasionally encountered findings in laboratory maintained rats of the strain and age employed.
HAEMATOLOGY
Statistically significant reductions in haemoglobin, haematocrit, mean
corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration were detected for 1000 mg/kg/day females when compared with controls.
No such changes were detected for 1000 mg/kg/day males or for animals of either sex treated at the remaining dose levels.
The statistically significant reductions in haemoglobin and MCH in 1000 and/or 250 mg/kg/day males were probably attributable to slightly higher than normal control values and, in the absence of effects on erythrocyte numbers and the fact that the majority of individual values were within the respective normal ranges, the intergroup differences were considered to be of no toxicological significance.

CLINICAL CHEMISTRY
A statistically significant reduction in plasma cholesterol was detected for both males and females treated with 1000 mg/kg/day when compared with controls. Many values from this and the other dose groups, including controls, were outside the respective normal ranges and, in the absence of any other changes in the blood chemical parameters measured, this finding was considered to be of dubious toxicological significance.
There were no other toxicologically significant changes in the blood chemical parameters measured.
The statistically significant increases in plasma albumin, albumin/globulin ratio and plasma calcium and sodium concentration all involved individual values within the respective normal ranges and, in isolation, all were regarded as incidental. The remaining intergroup differences were confined to a reduction in male 50 and 250 mg/kg/day alkaline phosphatase and female 1000 mg/kg/day alanine aminotransferase but reductions in these parameters cannot be regarded as toxicologically significant.

URINALYSIS
No data.

NEUROBEHAVIOUR
Behavioural Assessments
There were no treatment-related changes in behaviour detected.

Functional Performance Tests
No data.

ORGAN WEIGHTS
Females treated with 1000 mg/kg/day showed a statistically significant increase in liver and kidney weight, both absolute and relative to bodyweight, when compared with controls. All individual relative liver weights and three kidney weights were outside the respective normal ranges for rats of the strain and age used. The effect extended to 250 mg/kg/day females with increases in relative liver and kidney weight detected although statistical significance was only achieved for liver weight at this dose level.
No adverse organ weight changes were detected for 1000 mg/kg/day males or for animals of either sex treated with 250 or 50 mg/kg/day.
An increase in male 1000 mg/kg/day relative brain weight was detected but, in view of the reduced terminal bodyweight seen in these animals and the absence of any histopathological correlates, the increase was considered to be incidental. Relative adrenal and ovary weights were statistically significantly elevated in 1000 mg/kg/day females compared to those of controls. The level of significance was minimal (p<0.05) and, in the absence of any histopathological evidence to suggest an adverse effect in these organs, the increases were considered to be without toxicological importance.
The remaining statistically significant intergroup differences detected for the male treatment groups were confined to absolute weights and, in the absence of any similar changes in the respective relative weights, were considered to be fortuitous.

GROSS PATHOLOGY:
No treatment-related macroscopic abnormalities were detected.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver: Five females treated with 1000 mg/kg/day showed centrilobular hepatocyte enlargement in relation to treatment (p < 0.05). Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature. The condition was not observed amongst males, or amongst females from the remaining dose levels.
Kidneys: Five females dosed at 1000 mg/kg/day and one control female exhibited epithelial hypertrophy of inner cortical tubules. Although not quite attaining statistical significance, this minimal change was considered to be related to treatment at 1000 mg/kg/day for females, but was not associated with any degenerative changes. Such an effect of treatment was not observed for males or for females from the 250 or 50 mg/kg/day dose groups.
Thyroids: An increased incidence of follicular cell hypertrophy (p < 0.05), occasionally with associated colloid depletion, was observed in relation to treatment for females dosed at 1000 mg/kg/day. A similar effect was not observed for males or amongst females from the remaining treatment levels.
lungs: The incidence of groups of alveolar macrophages was increased in relation to treatment for females treated with 1000 rng/kg/day (pMesenteric lymph nodes: Sinus histiocytosis was observed for animals of either sex treated with 1000 mg/kg/day (p < 0.001 1. Histiocytes were generally foamy in appearance. Other lymphoid tissues including spleen, thymus, cervical lymph nodes, and Peyer’s patches were not affected and the condition did not involve animals from the remaining treatment groups.
Uterus: Although the incidence of uterine dilatation was increased for females dosed at 1000 mg/kg/day (p
HISTOPATHOLOGY: NEOPLASTIC (if applicable): NA.
HISTORICAL CONTROL DATA (if applicable): NA.
OTHER FINDINGS

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Justification please see Attachment file named "EC 294-785-9 NOAEL after 90d oral tx.pdf"

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No changes detected in any animal at this dose level.

Critical effects observed:

not specified

DISCUSSION

The administration of the test item by oral gavage for a period of ninety consecutive days resulted in treatment-related changes at dose levels of 1000 and 250 mg/kg/day.

A slight reduction in bodyweight development and food consumption and efficiency were observed for 1000 mg/kg/day males during the study but these were transient changes and did not persist past Week 10. No adverse effect on bodyweight gain or dietary intake was detected for 1000 mg/kg/day females, but haematological investigations revealed a mild hypochromic anaemia in this sex at this dose level. Absolute and relative liver weight were elevated for 1000 mg/kg/day females and microscopic examination of liver sections revealed changes identified as centrilobular hepatocyte enlargement for five out of the ten females treated at this dose level. This morphological change is often seen in the rodent liver following treatment with xenobiotics and is associated with induction of microsomal enzymes. In the absence of any associated degenerative or inflammatory changes, this is considered to be an adaptive response. Microscopic examination of thyroid sections revealed an increased incidence of follicular cell hypertrophy, occasionally with associated colloid depletion, for 1000 mg/kg/day females. One possible explanation for these changes is a secondary response to the changes occurring in the liver. It is possible that glucuronyltransferase may have been induced in response to treatment, thereby increasing thyroxine excretion and stimulating a compensatory thyroid response. There were no convincing changes in the blood chemical paramters measured, although plasma cholesterol was slightly reduced for animals of either sex treated with 1000 mg/kg/day.

 

Further treatment-related changes were also seen in females with increases in both absolute and relative kidney weight detected at 1000 mg/kg/day. Histopathologically five females exhibited epithelial hypertrophy of inner cortical tubules but there were no other findings indicative of an adverse effect on renal function at this dose level. Microscopic lung changes were also observed, identified as an increased incidence of alveolar macrophages and microscopic examination of mesenteric lymph nodes revealed histiocytosis at 1000 mg/kg/day. It is unclear whether these findings are related but the histiocytosis was probably a normal physiological consequence of infiltrating macrophages being unable to degrade their phagocytosed contents. There were no similar'findings for the other lymphatic tissues examined.

 

Treatment-related changes extended into the 250 mg/kg/day dose group with females showing a dose-related increase in relative liver and kidney weight. Although statistical significance was either absent or minimal (p < 0.05), individual values were raised compared with controls. In the absence of any histopathological correlates, however, this was considered not to represent an adverse health effect. No treatment-related changes were detected in any of the parameters measured for 250 mg/kg/day males or for animals of either sex treated with 50 mg/kg/day.

Conclusions:

Treatment-related changes were observed in all animals at the 1000 mg/kg bw/day dose level. Effects noted in females at the 250 mg/kg bw/day dose level were considered not to be adverse therefore the NOAEL is considered to be 250 mg/kg bw/day. The NOEL for both sexes is 50 mg/kg bw/day.

Executive summary:

Test Guidance

OECD 408 Guideline study

Method and Material

Rats (10 per sex per dose) were dosed by oral gavage for 90 consecutive days at dose levels of 0 (control: vehicle), 50, 250 and 1000 mg/kg bw/day test material in arachis oil. Clinical signs, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Opthalmoscopic examination was also performed on control group and high dose animals. All animals were subject to gross necropsy examination and a comprehensive histopathological evaluation of tissues was performed.

Results

Oral administration of the test material to rats for a period of ninety consecutive days at dose levels of up to 1000 mg/kg/day resulted in treatment-related changes at 1000 amongst animals of either sex and at 250 mg/kg/day amongst females only. No such changes were detected in animals treated with 50 mg/kg/day.

Conclusion

The "No Observed Effect Level" (NOEL) was considered to be 50 mg/kg/day. However, effects detected at 250 mg/kg/day were confined to marginal liver and kidney weight increases amongst females only and in the absence of any supporting histopathological correlates, these were considered not to represent an adverse health effect. The NOAEL was suggested to be 250 mg/kg/day.

 

Reference 2

Endpoint:

repeated dose toxicity: oral

Remarks:

other: One Generation Reproduction Study In The Rat

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 July 1998 to 22 December 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Section 4: Health Effects, Test Guidelines No. 415, 26 May 1983.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 ~ 7 weeks old
- Weight at study initiation: At the start of treatment the males weighed 158 to 205g, the females weighed 170 to 210g.
- Fasting period before study: none.
- Housing: Upon arrival, the animals were housed in groups of four in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays. During the mating period animals were transferred to a similar type cage on a one male to one female per cage basis.
- Water: tap-water was made available ad libitum.
- Acclimation period: at least 16 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21± 2
- Humidity (%): 55 ± 15
- Air changes: at least 15 air changes per hour
- Photoperiod: 12h light/12h dark light cycle

IN-LIFE DATES: From 28/07/98 - 22/12/98

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability of the test material formulations was determined by Safepharm Analytical Laboratory. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4C in the dark.
Samples were taken of each test material formulation and were analysed for concentration of the test substance at Safepharm Analytical Laboratory. The results indicate that the prepared formulations were within 10% of the nominal concentration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): NA.
- Concentration in vehicle: 0, 25, 125, 500 mg/mL
- Amount of vehicle (if gavage): 2mL/kg
- Lot/batch no. (if required): NA.
- Purity: NA.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC using an external standard.

Samples and standards prepared in methanol to give a concentration of 0.5 mg/mL

HPLC system: Hewlett Packard 1050
Column: Hypersil C18 (150 x 4.6 id)
Mobile phase: Eluent A - water and Eluent B - methanol
At time 0 - 10% B; 14 mins - 100% B and 26 mins - 100% B
Flowrate: 1.5 mL/min
UV detector wavelength: 240 nm
Injection volume: 25 µL
Retention time: 14 to 15 mins

Stability determination: analysed initally and after 14 days in the dark at +4°C.
Stability: 92-96% of initial concentration after 14 days

Formulation concentrations: sampled and analysed within three days of preparation at weekly intervals throughout study period.
Formulation: 93 - 110% of nominal

Duration of treatment / exposure:

Males: 10 weeks pre-mating, 16 days mating and through post-mating until weaning (Day 21 of weaning) - Total 16 weeks
Females 2 weeks pre-mating, 16 days mating and through post-mating (23 days) until weaning (Day 21 of weaning) - Total 11 weeks

Frequency of treatment:

The test material was administered once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24 animals/sex/dose/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of previous toxicology studies. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material, and the results of the study are believed to be of value in predicting the likely toxicity of the test material to man.

- Rationale for animal assignment (if not random): The animals were allocated to dose groups using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups.
- Rationale for selecting satellite groups: NA.
- Post-exposure recovery period in satellite groups: NDA.
- Section schedule rationale (if not random): Random

Positive control:

Not used

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked twice daily during the normal working week and once daily on weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily, immediately before, immediately after and one hour after dosing, for clinical signs of toxicity..

BODY WEIGHT: Yes
- Time schedule for examinations: During the maturation and mating period the parental generation animals were weighed weekly. Following mating the parental males were weighed weekly until termination. Parental generation females showing evidence of mating were weighed on Days 1, 4, 7, 14 and 21 post coitum. Parental generation females with a live litter were weighed on Days 1, 4, 7, 14 and 21 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. During the maturation periods food consumption was recorded weekly for each cage of parental generation adults. For parental generation females showing evidence of mating, food consumption was recorded for the periods covering Days 1 to 7, 7 to 14 and 14 to 21 post coiturn. For parental generation females with live litters, food consumption was recorded for the periods covering Days 1 to 7 and 7 to 14 post parturn.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No.

FOOD EFFICIENCY: Food Conversion Ratio was calculated weekly during the maturation period of the Parental generation.
Food Conversion Ratio = (Group mean bodyweight gain g/day) during week)/ Group mean food consumption g/rat/day)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: No.

CLINICAL CHEMISTRY: No

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin:
Testes with epididymides
Prostate
Seminal vesicles
Coagulating gland
Uterus with cervix
Ovaries
Pituitary
Liver
Kidneys

Other examinations:

This was a one-generation study, so parameters for F0 reproduction and F1 development were examined. Details are present in Reproduction and Development Endpoints.

Statistics:

The following parameters were analysed statistically, where appropriate using the test methods outlined as follows:

Adult male and female bodyweight during the maturation, gestation and lactation periods, adult male food consumption, female food consumption during maturation, or gestation and lactation. Values were analysed to establish homogeneity of group variances using Levene’s test followed by one-way analysis of variance. If the variances were unequal subsequent comparisons between control and treated groups were performed using Dunnett’s T3 Multiple Comparison Method. If variances were equal subsequent comparisons between control and treated groups were performed using Dunnett’s Multiple Comparison
Method.

Adult pre-coital intervals, female gestation lengths, offspring reflexological responses and litter sex ratios. Individual values were compared using the Kruskal-Wallis non-parametric rank sum test. Where significant differences were seen, pairwise comparison of control values against treated group values was performed using Mann-Whitney "U" test.

Histopathology
Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed conditions.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no toxicologically significant clinical findings throughout the study.

Mortality:
There were no unscheduled deaths that could be attributed to test material toxicity. There were several deaths that were considered to be due to dosing trauma.

BODY WEIGHT AND WEIGHT GAIN
Maturation
- At 1000 mg/kg there was a slight reduction in male bodyweight gain from Week 5 until the end of maturation phase compared to controls. Although male group mean bodyweights were slightly lower than control values the differences were not significant statistically. Female bodyweight
gain was not affected by treatment at this dose level.
- At 250 and 50 mg/kg/d there were no significant differences in male and female bodyweight gain throughout the respective maturation phases.

Post Maturation
At 1000 mg/kg/d male group mean bodyweight gain post maturation was slightly lower than that of controls, although the intergroup difference was not significant statistically.
At 250 and 50 mg/kg/d there were no significant differences in male bodyweight throughout this period.

Gestation
There were no significant intergroup differences in bodyweight gain for
females throughout the gestation period.

Lactation
There were no significant intergroup differences in bodyweight gain for females throughout the lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
There were no toxicologically significant effects.

WATER CONSUMPTION
No data.

OPHTHALMOSCOPIC EXAMINATION:
No data.

HAEMATOLOGY
No data.

NEUROBEHAVIOUR
Behavioural Assessments
There were no treatment-related changes in behaviour detected.

Functional Performance Tests
No data.

ORGAN WEIGHTS
At 1000 mg/kg/d there was a slight increase in male liver and kidney weight which, as a percentage of bodyweight, showed a statistically significant (p< 0.01) difference compared to controls. There was also a reduction in absolute prostate weight and prostate weight relative to bodyweight with the intergroup difference again achieving statistical significance compared to controls (p<0.05). Female liver and kidney weights (absolute and relative to bodyweight) were slightly increased compared to control values although only the increased kidney weight achieved statistical significance (p < 0.05).
At 250 and 50 mg/kg/d there were no significant differences compared to controls.

GROSS PATHOLOGY:
At terminal necropsy the incidence and distribution of macroscopic post rnortern findings show no treatment related trends. The gross abnormalities are those commonly observed in this study type.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no significant treatment related findings from the selected reproduction organs examined.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): NA.
HISTORICAL CONTROL DATA (if applicable): NA.
OTHER FINDINGS

Dose descriptor:

NOEL

Remarks:

adult toxicity

Effect level:

ca. 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

At 1000 mg/kg bw/day there was evidence of minor systemic toxicity during the in-life phase of the study. Male rats showed a slight reduction in bodyweight gain and animals of either sex showed slightly elevated liver and kidney weight compared to controls. No associated macroscopic or histological abnormalities were identified however. There were a number of unscheduled deaths at this dose level during the course of the study. These were all attributed to dosing trauma and were considered not to be indicative of test material toxicity.

Conclusions:

The administration of the test material to adult male and female rats prior to mating and throughout the reproductive cycle resulted in no effects on reproduction that could be attributed to the test material. There was evidence of minor systemic toxicity at a dose level of 1000 mg/kg/d. The NOEL for adult toxicity was 250 mg/kg/d, NOAEL was 1000 mg/kg/d

Executive summary:

TEST GUIDANCE

The study was designed to investigate the effects of the test material on the growth and reproductive performance of the rat and complies with OECD Guidelines for Testing of Chemicals, Section 4: Health Effects, Test Guidelines No. 415, 26 May 1983.

 

METHODS

The test material was administered orally, by gavage, to groups of twenty-four male and twenty-four female rats throughout maturation, mating, gestation and lactation. The dose levels were 50, 250 and 1000 mg/kg bw/day of test material with a similar sized control group receiving vehicle alone.

Following at least ten and two weeks of dosing respectively, male and female rats were paired within their dose groups to produce litters. At weaning of the offspring, all surviving animals were killed and examined macroscopically.

Parental animals were observed daily for clinical signs. Bodyweights and food consumption were recorded weekly during the maturation phase which was continued for males after the mating phase. Mated females were weighed and food consumption recorded on specific days post-coitum and post-partum.

 

Postmortem macroscopic examinations were performed on all adults and offspring, including decedents. At necropsy of adult males a semen sample was collected from the vas deferens of the left testis for sperm evaluation. Reproductive and potential target organs and any significant abnormalities from all parental animals were preserved in fixative. Histopathology was carried out on reproductive organs from control and high dose group parental animals. Additional testicular histopathology, involving staging of testicular spermatogenesis, was performed on control and high dose males.

 

RESULTS

At 1000 mg/kg bw/day there was evidence of minor systemic toxicity. Males showed slightly reduced bodyweight gain compared with that of controls during the maturation phase. In addition, there was a slight increase in male and female kidney weight at this dose level, a slight increase in male liver weight and prostrate weight was slightly reduced. There were no associated histopathological changes. There were no toxicologically significant findings at the remaining dose levels, although there were a number of unscheduled deaths at dose levels of 250 and 1000 mg/kg bw/day. These were attributable to dosing trauma and were not related to test material toxicity.

 

CONCLUSION

The administration of the test material to adult male and female rats throughout the reproductive cycle resulted in no effects on reproduction that could be attributed to the test material. There was evidence of minor systemic toxicity at a dose level of 1000 mg/kg bw/day. The NOEL for adult toxicity was 250 mg/kg bw/day, NOAEL was 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

562 µg/cm²

Study duration:

subacute

Species:

rat

Additional information

Oral route

Key study

The investigation was performed in accordance with OECD 408. Rats (10 per sex per dose) were dosed by oral gavage for 90 consecutive days at dose levels of 0 (control: vehicle), 50, 250 and 1000 mg/kg bw/day test material in arachis oil. Clinical signs, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Opthalmoscopic examination was also performed on control group and high dose animals. All animals were subject to gross necropsy examination and a comprehensive histopathological evaluation of tissues was performed.

Oral administration of the test material to rats for a period of ninety consecutive days at dose levels of up to 1000 mg/kg/day resulted in treatment-related changes at 1000 amongst animals of either sex and at 250 mg/kg/day amongst females only. No such changes were detected in animals treated with 50 mg/kg/day.

The "No Observed Effect Level" (NOEL) was considered to be 50 mg/kg/day. However, effects detected at 250 mg/kg/day were confined to marginal liver and kidney weight increases amongst females only and in the absence of any supporting histopathological correlates, these were considered not to represent an adverse health effect. The NOAEL was suggested to be 250 mg/kg/day.

Supporting study

The supporting study was designed to investigate the effects of the test material on the growth and reproductive performance of the rat and complies with OECD Guidelines for Testing of Chemicals, Section 4: Health Effects, Test Guidelines No. 415, 26 May 1983.

The test material was administered orally, by gavage, to groups of twenty-four male and twenty-four female rats throughout maturation, mating, gestation and lactation. The dose levels were 50, 250 and 1000 mg/kg bw/day of test material with a similar sized control group receiving vehicle alone.

Following at least ten and two weeks of dosing respectively, male and female rats were paired within their dose groups to produce litters. At weaning of the offspring, all surviving animals were killed and examined macroscopically.

Parental animals were observed daily for clinical signs. Bodyweights and food consumption were recorded weekly during the maturation phase which was continued for males after the mating phase. Mated females were weighed and food consumption recorded on specific days post-coitum and post-partum.

The offspring were observed daily for clinical signs. The litter signs and individual pup bodyweights were recorded on specific days post-partum. During the lactation period the offspring were observed for intra-litter onset and duration of landmarks of physical development. On specific days of lactation, reflexological assessment of offspring was performed.

Postmortem macroscopic examinations were performed on all adults and offspring, including decedents. At necropsy of adult males a semen sample was collected from the vas deferens of the left testis for sperm evaluation. Reproductive and potential target organs and any significant abnormalities from all parental animals were preserved in fixative. Histopathology was carried out on reproductive organs from control and high dose group parental animals. Additional testicular histopathology, involving staging of testicular spermatogenesis, was performed on control and high dose males.

Reproduction was unaffected by treatment. The incidence of non-treatment related total litter losses seen at 1000 mg/kg bw/day and 50 mg/kg bw/day is a recognised aberration with the particular strain and source of rat used in this study.

At 1000 mg/kg bw/day there was evidence of minor systemic toxicity. Males showed slightly reduced bodyweight gain compared with that of controls during the maturation phase. In addition, there was a slight increase in male and female kidney weight at this dose level, a slight increase in male liver weight and prostrate weight was slightly reduced. There were no associated histopathological changes. There were no toxicologically significant findings at the remaining dose levels, although there were a number of unscheduled deaths at dose levels of 250 and 1000 mg/kg bw/day. These were attributable to dosing trauma and were not related to test material toxicity.

The administration of the test material to adult male and female rats throughout the reproductive cycle resulted in no effects on reproduction that could be attributed to the test material. There was evidence of minor systemic toxicity at a dose level of 1000 mg/kg bw/day. The No Observed Effect Level for reproductive/developmental effects was 1000 mg/kg bw/day and the No Observed Effect Level for adult toxicity was 250 mg/kg bw/day (A NOAEL of 1000 mg/kg bw/d was proposed since only minor systemic effects were observed at this dose level).

Inhalation route

The substance has been shown to have a high onset boiling point (decomposition takes place from 324°C at 101.325 kPa) and the vapour pressure has been determined to be3.5 x 10E-04Pa at 25 °C. It is therefore expected that inhalation exposure will be low under general use conditions at ambient temperature. Results from investigation of systemic toxicity via repeated dose oral administration of the test material suggest that the determined vapour pressure of 0.05 Pa at 70 °C also gives no cause for concern. The inhalation route is therefore not the most applicable method of investigating repeated dose toxicity.

Dermal route

The 28-day repeat dose dermal toxicity study was performed according to OECD TG 410. The test material was applied to the shaved dorsal area and sides of rats (5 animals/sex/dose) at dose levels of 0, 100, 300 or 1000 mg/kg bw/day, five days/week for four weeks. After application of the test material the dose site was covered with a semi-occlusive dressing for 6 hours, after which the dressing was removed and the skin wiped free of any excess test material. The effect of the test material on the rats was evaluated according to physical appearance, dermal irritation, body weight, food consumption, haematology, clinical chemistry, organ weights, gross and microscopic pathology.

There were no mortalities and no evidence of systemic toxicity was seen in any of the groups during the study. Evidence of mild dermal irritation (erythema after the first week of dosing and/or desquamation during the third and fourth week) was seen in two females at 100 mg/kg bw/day, one male and most females at 300 mg/kg bw/day and most animals at 1000 mg/kg bw/day. No severe dermal irritation or evidence of deep tissue damage was apparent, however. Microscopic examination of treated skin from control and high dose animals revealed minimal to slight epidermal hyperplasia in several (6 of 9) animals in the 1000 mg/kg bw/day group. Evaluations of body weights, food consumption, clinical chemistry studies, organ weights, organ/body weight ratios and microscopic examination of liver and kidneys from animals in the 1000 mg/kg bw/day group did not reveal any effects considered to be related to administration of the test material. The NOEL for systemic toxicity was determined to be 1000 mg/kg bw/day. The LOAEL for local effects was 100 mg/kg bw/day based on mild skin irritation observed in 1/5 females.

Justification for classification or non-classification

According to ECHA Guidance on the Application of the CLP Criteria (Version 5.0; July 2017), classification as STOT RE 2 is applicable when significant toxic effects observed in a 90-day repeated dose study conducted in experimental animals are seen to occur within the guidance value range of 10 to ≤ 100 mg/kg bw/day. The NOAEL value from the 90-day repeated dose oral study in rats is 250 mg/kg b/day and therefore lies outside the range leading to classification.