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Description of key information

The oral administration of 4,4’-Isopropylidenediphenol,oligomeric reaction products with 1-chloro-2,3-expoxypropane, reaction products with 2-methylimidazole to rats by gavage, at dose levels of 25, 100 and 400 mg/kg bw/day, resulted in treatment-related findings in animals of either sex treated with 400 and 100 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was considered to be 25 mg/kg bw/day for both males and females.

The stomach changes identified at 400 mg/kg bw/day and the effects on body weight development and/or food intake in animals from the 400 or 100 mg/kg bw/day dose groups were considered to be a result of local irritation of the test item rather than a consequence of systemic toxicity.

Microscopic changes in the lymph node from animals of either sex given 400 or 100 mg/kg bw/day were considered to be adverse. Whilst microscopic changes were observed in the lymph nodes at 100 mg/kg bw/d in both sexes, this was in the absence of abscess formation and therefore considered to have had no functional impact and unlikely to be considered adverse.Taking into account the overall results a No Observed Adverse Effect Level (NOAEL) can be established at 100 mg/kg bw/day for either sex.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for seven days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 219 to 259g, the females weighed 171 to 200g, and were approximately six to eight weeks old.
Sex:
male/female
Details on test animals or test system and environmental conditions:
The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 5; expiry date for the batch number 070715MA was extended to 07 July 2016. Mains drinking water was supplied from polycarbonate bottles attached to the cage. 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 were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.
Vehicle:
arachis oil
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A summary incidence of daily clinical observations is given in Table 1. Individual data are presented in Appendix 1.

Animals of either sex treated with 400 mg/kg bw/day showed instances of increased post dose salivation from Week 1 and 2 of treatment for males and females respectively. At 100 mg/kg bw/day, animals of either sex showed isolated instances of increased post dose salivation between Days 74 and 76 (males) and on Day 81 (females). Observations of this nature are commonly observed following the oral administration of an unpalatable or irritant test item and as such are considered not to represent systemic toxicity.

One control female showed signs of hunched posture on Day 76 and pallor of the extremities on Days 77 and 78. In isolation this was considered to be an incidental finding.

There were no clinical signs for any of the animals receiving the test item at a dose level of 25 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean weekly body weights and standard deviations are given in Table 6 and are presented graphically in Figure 1 and Figure 2. Group mean weekly body weight gains and standard deviations are given in Table 7 (statistically significant differences are indicated). Individual data are given in Appendix 7 and Appendix 8.

At 400 mg/kg bw/day males generally showed lower mean body weight gains when compared with controls, attaining statistical significance during Weeks 4, 5, 8 and 11. Males treated with 100 mg/kg bw/day also showed statistically significantly lower body weight gains during Weeks 4 and 11. Overall body weight gains in males treated with 400 or 100 mg/kg bw/day were approximately 17% and 9% lower than controls, respectively.

Females treated with 400 or 100 mg/kg bw/day showed a statistically significant reduction in body weight gain during the first week of treatment with subsequent recovery evident. Fluctuations were noted during the latter stages of the study, achieving statistical significance in some cases taking into account the minimal effect on overall body weight gain and the lack of a true dose related response, the intergroup differences were considered not to be of toxicological significance.

Body weight gains for animals of either sex treated with 25 mg/kg bw/day were generally similar to controls throughout the treatment period. A statistically significant lower body weight gain for males during Week 11 was evident however in isolation and in the absence of any effect on overall body weight gain, this was considered to be incidental. A statistically significant increase in body weight gain was also evident in females from this treatment group during Week 12 however an increase in body weight gain is generally considered not to be of toxicological importance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Group Mean Food Consumptions are given in Table 8 and are presented graphically in Figure 3 and Figure 4. Weekly food efficiencies are given in Table 9.

Overall food consumption for males treated with 400 and 100 mg/kg bw/day was slightly lower (10% and 8% respectively) when compared to controls. No such effects were evident in females treated with 400 or 100 mg/kg bw/day or in animals of either sex treated with 25 mg/kg bw/day.

No overall adverse effect on food conversion efficiency was evident in treated animals. Minor fluctuations were evident in treated animals however these reflected minor differences in body weight gains and/or food intake.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Individual ophthalmoscopic examination findings are given in Appendix 9.

There were no treatment-related ocular effects detected.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 400 mg/kg bw/day females showed a statistically significant reduction in MCH, MCV and activated partial thromboplastin time when compared to controls. Individual values for MCH and MCV exceeding the background control ranges were limited to single incidences. However, these changes correlate with the increased haematopoiesis observed in the spleens of these females. Whilst it could be argued that an association with treatment cannot be excluded, high dose males presented comparable haematological changes in the absence of any associated spleen histopathology. The mild severity in spleen response in high dose females may be due to individual variation and unlikely to be of significance.

Males treated with 400 mg/kg bw/day showed a statistically significant reduction in levels of haemoglobin (Hb), haematocrit (Hct), mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) when compared to controls. The majority of the individual values for these parameters were within the background control ranges. Individual values for MCH and MCV exceeding the background control ranges were limited to single incidences. At this dose level males also showed a statistically significant increase in total leucocyte count, neutrophils, lymphocytes and platelet count when compared to controls. With the exception of platelet count, the majority of the individual values were above the background control ranges. Accompanying histopathological changes (sinus histiocytosis) were associated with the increases in nucleated white blood counts.
Description (incidence and severity):
Group mean values and standard deviations for test and control group animals are given in Table 11 (statistically significant differences are indicated). Individual data are given in Appendix 12 and Appendix 13.

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

At 400 mg/kg bw/day males showed a statistically significant reduction in albumin and urea. The effect on urea also extended to males treated with 100 mg/kg bw/day. The majority of individual values for albumin were below the background control ranges however the majority of values for urea were within the background control ranges. Males treated with 400 mg/kg bw/day also showed a statistically significant increase in potassium and aspartate aminotransferase. All individual values for potassium were within the background control ranges however the majority of individual values for aspartate aminotransferase were above the background control ranges. These findings were not present in the corresponding females. In the absence of any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.

Females treated with 400 mg/kg bw/day showed a statistically significant increase in albumin/globulin ratio and a statistically significant reduction in alkaline phosphatase. All of the individual values were within background control ranges for both parameters and in the absence of any histopathological correlates, the intergroup differences were considered not to be of toxicological importance.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional Observations
A summary incidence of behavioral assessment observations is given in Table 2 and group mean behavioral assessment scores are given in Table 3. Individual values are given in Appendix 2 and Appendix 3. Group mean functional performance test values and standard deviations are given in Table 4. Individual values are given in Appendix 4 and Appendix 5. Group mean sensory reactivity assessments are given in Table 5. Individual responses are given in Appendix 6.

Behavioral Assessments
There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests
There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Group mean absolute and relative organ weights and standard deviations for test and control group animals are presented in Table 13 (statistically significant differences are indicated). Individual data are given in Appendix 15 and Appendix 16.

There were no toxicologically significant effects detected in the organ weights measured.

Females treated with 400 or 100 mg/kg bw/day showed statistically significant reductions in heart weight both absolute and relative to terminal body weight. The majority of individual values were within the historical background control ranges and as there were no histopathological correlates, these intergroup differences were considered to be of no toxicological importance.

Males treated with 25 mg/kg bw/day showed a statistically significant reduction in adrenal weights both absolute and relative to terminal body weight. Females from this treatment group also showed a statistically significant reduction in absolute and relative kidney weights. The majority of individual values were within historical background control ranges and there were no histopathological correlates or a similar effect at 400 or 100 mg/kg bw/day. Therefore these intergroup differences were considered to be incidental.
Gross pathological findings:
no effects observed
Description (incidence and severity):
A summary incidence of necropsy findings is given in Table 12. Individual data are given in Appendix 14.

At 400 mg/kg bw/day the majority of males and a small number of females showed thickening of the stomach, raised limiting ridge and/or raised white patches in the nonglandular region of the stomach. No such effects were detected in animals of either sex treated with 100 or 25 mg/kg bw/day. These findings were consistent with the irritant nature of the test item as confirmed by the histopathological findings in the stomach.

One male each from the 400 and 100 mg/kg bw/day dose groups had increased pelvic space in the kidney (left and right, respectively) with the 100 mg/kg bw/day male also showing fluid filled kidneys. In the absence of any histopathological correlates these findings were considered to be incidental.

Animals of either sex across all dose levels including controls showed reddened lungs. In the absence of any histopathological correlates these findings were considered to be incidental.

One male treated 100 mg/kg bw/day had a small left seminal vesicle, one control male had an enlarged heart and a further control male had an enlarged spleen. In the absence of any histopathological correlates these findings were considered to be incidental.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A complete histopathology phase report is presented in Annex 1.

The following treatment related microscopic abnormalities were detected:

Mesenteric Lymph Node
Histiocyte aggregates were present in the sinusoids, in the mesenteric lymph node of all the 400 mg/kg bw/day animals at a mild or moderate severity level with abscess formation also noted in seven males and one female from this dose level. Histiocyte aggregates were also present to minimal or mild severity in nine males and seven females at 100 mg/kg bw/day. At 25 mg/kg bw/day there were no notable findings.

Spleen
Increased hematopoiesis (mild severity) was noted in the spleen of one, two, three and five females from control, 25, 100 and 400 mg/kg bw/day, respectively. Such findings were not evident in males from these dose groups.

Stomach
Changes to the stomach including hyperplasia of the non-glandular region or limiting ridge, ulceration and inflammatory cell infiltration, also in the non-glandular area, were present in most males and females in the 400 mg/kg bw/day dose group. These changes correlated with macroscopic findings noted at necropsy.

Inflammatory infiltrate in the glandular area was present in two males from the 400 mg/kg bw/day dose group, one of which had ulceration of the non-glandular region and this may have been an associated change rather than a direct effect of the test item.

Thyroid Gland
Minimal hypertrophy of the follicular cells was present in two, four, four and six males from control, 25, 100 and 400 mg/kg bw/day dose groups, respectively. In the corresponding females minimal hypertrophy of the follicular cells was present in one, one, two and eight females from control, 25, 100 and 400 mg/kg bw/day dose groups, respectively.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
The oral administration of 4,4’-Isopropylidenediphenol,oligomeric reaction products with 1-chloro-2,3-expoxypropane, reaction products with 2-methylimidazole to rats by gavage, at dose levels of 25, 100 and 400 mg/kg bw/day, resulted in treatment-related findings in animals of either sex treated with 400 and 100 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was considered to be 25 mg/kg bw/day for both males and females.

The stomach changes identified at 400 mg/kg bw/day and the effects on body weight development and/or food intake in animals from the 400 or 100 mg/kg bw/day dose groups were considered to be a result of local irritation of the test item rather than a consequence of systemic toxicity.

Microscopic changes in the lymph node from animals of either sex given 400 or 100 mg/kg bw/day were considered to be adverse. Whilst microscopic changes were observed in the lymph nodes at 100 mg/kg bw/d in both sexes, this was in the absence of abscess formation and therefore considered to have had no functional impact and unlikely to be considered adverse.Taking into account the overall results a No Observed Adverse Effect Level (NOAEL) can be established at 100 mg/kg bw/day for either sex.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guideline:

The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 25, 100 and 400 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, body weight 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.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Mortality

There were no unscheduled deaths.

Clinical Observations

Animals of either sex treated with 400 mg/kg bw/day showed instances of increased post dose salivation from Week 1 (males) and Week 2 (females) onwards. At 100 mg/kg bw/day animals of either sex showed isolated instances of increased post dose salivation during the latter stage of the study. No such effects were evident in animals of either sex treated with 25 mg/kg bw/day.

Behavioral Assessment

There were no treatment-related changes in behavioral parameters measured.

Functional Performance Tests

There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

Overall body weight gains for males receiving the test item at 400 or 100 mg/kg bw/day showed a dose related reduction when compared to controls. Generally males treated with 400 mg/kg bw/day showed lower weekly gains during the treatment period.

Females receiving the test item at 100 or 400 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment, however improvement was evident thereafter.

Animals of either sex treated with 25 mg/kg bw/day generally showed similar body weight gains as controls.

Food Consumption

Males treated with 400 or 100 mg/kg bw/day showed slightly reduced overall food consumption. No such effects were detected in females treated with 400 or 100 mg/kg bw/day or in animals of either sex treated with 25 mg/kg bw/day.

Water Consumption

There were no treatment-related effects detected in water consumption.

Ophthalmoscopy

There were no treatment-related ocular effects detected.

Hematology

At 400 mg/kg bw/day females showed statistically significant reductions in mean corpuscular haemoglobin, mean corpuscular volume and a statistically significant increase in neutrophil count when compared to controls. These changes correlated with the histopathological findings evident in the spleen. The corresponding males showed a similar response, but revealed no histopathological correlates.

Blood Chemistry

There were no toxicologically significant effects on the blood chemical parameters measured.

Necropsy

At 400 mg/kg bw/day four males and two females showed thickening of the stomach, raised white patches on the glandular region of the stomach and a raised limiting ridge. One male showed thickening of the stomach and a raised limiting ridge and another male showed raised white patches on the non glandular region of the stomach and a raised limiting ridge. A further male and female from this treatment group showed raised white patches on the nonglandular region, and another male showed only thickening of the stomach. No such effects were detected in animals of either sex treated with 100 or 25 mg/kg bw/day.

Organ Weights

There were no toxicologically significant effects detected in the organ weights measured.

Histopathology

The following treatment related microscopic abnormalities were detected:

Mesenteric Lymph Node

Histiocyte aggregates were present in the sinusoids, in the mesenteric lymph node of all the high dose animals at a mild or moderate severity level. There were also abscess formation noted in seven males and one female at 400 mg/kg bw/day. Histiocyte aggregates was present to minimal or mild severity in nine males and seven females at 100 mg/kg bw/day and at 25 mg/kg bw/day were within expected limits.

Spleen

There was increased hematopoiesis present in the spleen of one, two, three and five females from control, 25, 100 and 400 mg/kg bw/day, respectively, of a mild severity.

Stomach

Changes to the stomach included hyperplasia of the non-glandular region or limiting ridge, ulceration and inflammatory cell infiltration, also in the non-glandular area, were present in most males and females in the 400 mg/kg bw/day dose group. These changes correlate with the findings noted at necropsy.

Inflammatory infiltrate in the glandular area was present in two males from the 400 mg/kg bw/day dose group, one of which had ulceration of the non-glandular region and this may have been an associated change rather than a direct effect of the test item.

Thyroid Gland

Minimal hypertrophy of the follicular cells was present in two, four, four and six males from control, 25, 100 and 400 mg/kg bw/day, respectively. In the corresponding females minimal hypertrophy of the follicular cells was present in one, one, two and eight females from control, 25, 100 and 400 mg/kg bw/day, respectively. As there were no weight increases or any associated liver hypertrophy, which may have indicated a disruption in thyroid hormone synthesis, these changes were considered unlikely to represent an adverse effect of treatment with the test item.

Conclusion

The oral administration of 4,4’-Isopropylidenediphenol,oligomeric reaction products with 1-chloro-2,3-expoxypropane, reaction products with 2-methylimidazole to rats by gavage, at dose levels of 25, 100 and 400 mg/kg bw/day, resulted in treatment-related findings in animals of either sex treated with 400 and 100 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was considered to be 25 mg/kg bw/day for both males and females.

The stomach changes identified at 400 mg/kg bw/day and the effects on body weight development and/or food intake in animals from the 400 or 100 mg/kg bw/day dose groups were considered to be a result of local irritation of the test item rather than a true effect of systemic toxicity.

Microscopic changes in the lymph node from animals of either sex given 400 or 100 mg/kg bw/day were considered to be adverse. Whilst microscopic changes were observed in the lymph nodes at 100 mg/kg bw/d in both sexes, this was in the absence of abscess formation and therefore considered to have had no functional impact and unlikely to be considered adverse. Taking into account the overall results a No Observed Adverse Effect Level (NOAEL) can be established at 100 mg/kg bw/day for either sex.

Endpoint conclusion
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

Justification for classification or non-classification