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Description of key information

No study is available to assess the repeated dose toxicity of (1-methylpropylidene)bis[tert-butyl] peroxide, therefore, it is evaluated through a read across to 2,2-di(tert-butylperoxy) butane (CAS # 2167-23-9).

A study was designed to investigate the systemic toxicity and potential adverse effects of Trigonox D-C50 (50.4% 2,2-di(tert-butylperoxy) butane (CAS # 2167-23-9) in isododecane) on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996). Trigonox D-C50 was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 150 and 500 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Adult males were terminated on Days 43 or 44, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no unscheduled deaths during the study. Animals of either sex treated with 500 mg/kg bw/day showed increased salivation from Day 4 onwards. Animals of either sex treated with 150 mg/kg bw/day also showed increased salivation albeit to a lesser extent. Two males treated with 500 mg/kg bw/day had pilo- erection between Days 20 and 31. No such effects were detected in animals of either sex treated with 50 mg/kg bw/day. One male treated with 500 mg/kg bw/day had pilo-erection during the final week of assessments. Another male showed increased salivation during Week 2 assessments. No such effects were evident in females treated with 500 mg/kg bw/day or in animals of either sex treated with 150 or 50 mg/kg bw/day. There was no effect of treatment with the test item at any dose level on functional performance in animals of either sex. Sensory reactivity scores across all test item-treated dose groups were similar to controls. Males treated with 500 mg/kg bw/day showed a reduction in body weight gain during the first two weeks of treatment. Overall body weight gain for these males was also reduced.  A slight reduction in body weight gain was evident in females treated with 500 mg/kg bw/day during the first week of treatment, however, recovery was evident thereafter. No adverse effects were evident in animals of either sex treated with 150 or 50 mg/kg bw/day. Males treated with 500 mg/kg bw/day showed a slight reduction in food consumption during the first two weeks of treatment. Recovery was evident thereafter. No adverse effects were evident in males treated with 150 or 50 mg/kg bw/day or in treated females during maturation, gestation or lactation. Food conversion efficiency was slightly reduced in animals of either sex treated with 500 mg/kg bw/day during the first week of treatment, however, recovery was evident thereafter. Males treated with 500 mg/kg bw/day showed an increase in overall water consumption during the pre-pairing phase. No such effects were evident in females treated with 500 mg/kg bw/day or in animals of either sex treated with 150 or 50 mg/kg bw/day.

There were no toxicologically significant effects detected in the hematological parameters examined. Animals of either sex treated with 500 mg/kg bw/day showed an increase in alanine aminotransferase. Males from this treatment group also showed an increase in aspartate aminotransferase. Females treated with 500 mg/kg bw/day also showed increases in glucose, total protein, albumin and cholesterol. The effect on total protein and cholesterol also extended to females treated with 150 mg/kg bw/day. No toxicologically significant effects were detected in males treated with 150 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day.Eleven males treated with 500 mg/kg bw/day had mottled kidneys. Eight of these males also had enlarged kidneys, one male also had increased pelvic space in the left kidney and the left kidney was fluid filled, one male had an enlarged and dark liver and another male had enlarged kidneys. Five males treated with 150 mg/kg bw/day had mottled kidneys and one of these males also had increased pelvic space in both kidneys. One male treated with 50 mg/kg bw/day also had mottled kidneys. No such effects were detected in treated females. Males from all treatment groups and females treated with 500 and 150 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weight. Males treated with 500 and 150 mg/kg bw/day also showed an increase in absolute and relative kidney weights. No such effects were evident in females treated with 50 mg/kg bw/day. The following treatment-related microscopic abnormalities were detected:

Kidneys: An increase in hyaline droplets (mild or moderate) was evident in all males treated with 500 and 150 mg/kg bw/day and in one male treated with 50 mg/kg bw/day (mild).Basophilic tubules were present in eleven males treated with 500 mg/kg bw/day and ten males treated with 150 mg/kg bw/day. In the remaining male treated with 500 mg/kg bw/day moderate nephropathy was apparent (tubular basophilia along with dilation and interstitial changes). Proteinaceous casts were also present (minimal to moderate) in all males treated with 500 mg/kg bw/day and in two males treated with 150 mg/kg bw/day.

Liver:Periportal pigment was present in the bile ducts of three males and two females treated with 500 mg/kg bw/day and in four males treated with 150 mg/kg bw/day. Periportal Kupffer cell pigmentation was present in four males and one female treated with 500 mg/kg bw/day and in one male treated with 150 mg/kg bw/day. Special staining carried out on one control and one male treated with 500 mg/kg bw/day was negative for bile pigment, lipofuscin and hemosiderin. Inflammatory cell infiltration in the periportal area was present in two males and one female treated with 500 mg/kg bw/day. Centrilobular hypertrophy, minimal or mild, was present in four males and four females treated with 500 mg/kg bw/day and in two males and one female treated with 150 mg/kg bw/day. No treatment related changes were evident in animals of either sex treated with 50 mg/kg bw/day.

Thyroid Gland: Minimal follicular cell hypertrophy was present in three males and two females treated with 500 mg/kg bw/day and in three males treated with 150 mg/kg bw/day. No treatment related changes were evident in animals of either sex treated with 50 mg/kg bw/day.

Centrilobular hypertrophy is generally considered to be due to an adaptive response to mixed function oxidase. This change results in the increased clearance of thyroid hormones and the subsequent compensatory increased production causes hypertrophy of the follicular cells (Capen C.C et al; 2002, Cattley R.C et al; 2002 and Zabka T.S et al; 2011).

The oral administration of

Trigonox D-C50 (50.4% 2,2-di(tert-butylperoxy) butane (CAS # 2167-23-9) in isododecane) to Wistar Han™:RccHan™:WIST strain rats for a period of up to seven weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels 50, 150 and 500 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 500 and 150 mg/kg bw/day. These included reduced initial body weight gains in either sex at 500 mg/kg bw/day, reduced food consumption and increased water consumption in males at 500 mg/kg bw/day, organ weight changes in either sex at 500 and 150 mg/kg bw/day, macroscopic changes in males at 500 and 150 mg/kg bw/day and microscopic changes in either sex at 500 and 150 mg/kg bw/day. A No Observed Effect Level (NOEL) for systemic toxicity was considered to be 50 mg/kg bw/day for either sex.

The inflammatory cell infiltration in the periportal area of the liver in either sex at 500 mg/kg bw/day was considered to represent an adverse effect of treatment. The microscopic liver changes evident in females treated with 150 mg/kg bw/day were considered to be an adaptive response to mixed function oxidase induction (Capen C.C et al; 2002, Cattley R.C et al; 2002 and Zabka T.S et al; 2011). Therefore, a No Observed Adverse Effect Level (NOAEL) can be established at 150 mg/kg bw/day for females. Although the kidney findings of tubular basophilia and proteinaceous casts in male kidneys could be considered an adverse effect, these findings were considered to be associated with alpha 2u-globulin and formation of hyaline droplets, an effect recognized as being both species and sex specific and not relevant for humans. In terms of risk assessment, these findings observed on this study would suggest that a No Observed Adverse Effect Level (NOAEL) can be established at 150 mg/kg bw/day for males because the findings do not reflect true systemic toxicity.

Therefore the NOAEL for the repeated dose toxicity was considered to be 150 mg/kg bw/day forTrigonox D-C50 which is equivalent to 75 mg/kg bw/day for pure 2,2-di(tert-butylperoxy) butane.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See attached document.
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Duration of treatment / exposure:
up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females
Frequency of treatment:
Daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
other: male rat specific alpha 2u-globulin nephropathie
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Executive summary:

A study was designed to investigate the systemic toxicity and potential adverse effects of Trigonox D-C50 (50.4% 2,2-di(tert-butylperoxy) butane(CAS # 2167-23-9) in isododecane) on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996). Trigonox D-C50 was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 150 and 500 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Adult males were terminated on Days 43 or 44, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no unscheduled deaths during the study. Animals of either sex treated with 500 mg/kg bw/day showed increased salivation from Day 4 onwards. Animals of either sex treated with 150 mg/kg bw/day also showed increased salivation albeit to a lesser extent. Two males treated with 500 mg/kg bw/day had pilo- erection between Days 20 and 31. No such effects were detected in animals of either sex treated with 50 mg/kg bw/day. One male treated with 500 mg/kg bw/day had pilo-erection during the final week of assessments. Another male showed increased salivation during Week 2 assessments. No such effects were evident in females treated with 500 mg/kg bw/day or in animals of either sex treated with 150 or 50 mg/kg bw/day. There was no effect of treatment with the test item at any dose level on functional performance in animals of either sex. Sensory reactivity scores across all test item-treated dose groups were similar to controls. Males treated with 500 mg/kg bw/day showed a reduction in body weight gain during the first two weeks of treatment. Overall body weight gain for these males was also reduced.  A slight reduction in body weight gain was evident in females treated with 500 mg/kg bw/day during the first week of treatment, however, recovery was evident thereafter. No adverse effects were evident in animals of either sex treated with 150 or 50 mg/kg bw/day. Males treated with 500 mg/kg bw/day showed a slight reduction in food consumption during the first two weeks of treatment. Recovery was evident thereafter. No adverse effects were evident in males treated with 150 or 50 mg/kg bw/day or in treated females during maturation, gestation or lactation. Food conversion efficiency was slightly reduced in animals of either sex treated with 500 mg/kg bw/day during the first week of treatment, however, recovery was evident thereafter. Males treated with 500 mg/kg bw/day showed an increase in overall water consumption during the pre-pairing phase. No such effects were evident in females treated with 500 mg/kg bw/day or in animals of either sex treated with 150 or 50 mg/kg bw/day.

There were no toxicologically significant effects detected in the hematological parameters examined. Animals of either sex treated with 500 mg/kg bw/day showed an increase in alanine aminotransferase. Males from this treatment group also showed an increase in aspartate aminotransferase. Females treated with 500 mg/kg bw/day also showed increases in glucose, total protein, albumin and cholesterol. The effect on total protein and cholesterol also extended to females treated with 150 mg/kg bw/day. No toxicologically significant effects were detected in males treated with 150 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day.Eleven males treated with 500 mg/kg bw/day had mottled kidneys. Eight of these males also had enlarged kidneys, one male also had increased pelvic space in the left kidney and the left kidney was fluid filled, one male had an enlarged and dark liver and another male had enlarged kidneys. Five males treated with 150 mg/kg bw/day had mottled kidneys and one of these males also had increased pelvic space in both kidneys. One male treated with 50 mg/kg bw/day also had mottled kidneys. No such effects were detected in treated females. Males from all treatment groups and females treated with 500 and 150 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weight. Males treated with 500 and 150 mg/kg bw/day also showed an increase in absolute and relative kidney weights. No such effects were evident in females treated with 50 mg/kg bw/day. The following treatment-related microscopic abnormalities were detected:

Kidneys:An increase in hyaline droplets (mild or moderate) was evident in all males treated with 500 and 150 mg/kg bw/day and in one male treated with 50 mg/kg bw/day (mild).Basophilic tubules were present in eleven males treated with 500 mg/kg bw/day and ten males treated with 150 mg/kg bw/day. In the remaining male treated with 500 mg/kg bw/day moderate nephropathy was apparent (tubular basophilia along with dilation and interstitial changes). Proteinaceous casts were also present (minimal to moderate) in all males treated with 500 mg/kg bw/day and in two males treated with 150 mg/kg bw/day.

Liver:Periportal pigment was present in the bile ducts of three males and two females treated with 500 mg/kg bw/day and in four males treated with 150 mg/kg bw/day. Periportal Kupffer cell pigmentation was present in four males and one female treated with 500 mg/kg bw/day and in one male treated with 150 mg/kg bw/day. Special staining carried out on one control and one male treated with 500 mg/kg bw/day was negative for bile pigment, lipofuscin and hemosiderin. Inflammatory cell infiltration in the periportal area was present in two males and one female treated with 500 mg/kg bw/day. Centrilobular hypertrophy, minimal or mild, was present in four males and four females treated with 500 mg/kg bw/day and in two males and one female treated with 150 mg/kg bw/day. No treatment related changes were evident in animals of either sex treated with 50 mg/kg bw/day.

Thyroid Gland: Minimal follicular cell hypertrophy was present in three males and two females treated with 500 mg/kg bw/day and in three males treated with 150 mg/kg bw/day. No treatment related changes were evident in animals of either sex treated with 50 mg/kg bw/day.

Centrilobular hypertrophy is generally considered to be due to an adaptive response to mixed function oxidase. This change results in the increased clearance of thyroid hormones and the subsequent compensatory increased production causes hypertrophy of the follicular cells (Capen C.C et al; 2002, Cattley R.C et al; 2002 and Zabka T.S et al; 2011).

The oral administration of

Trigonox D-C50 (50.4% 2,2-di(tert-butylperoxy) butane (CAS # 2167-23-9) in isododecane) to Wistar Han™:RccHan™:WIST strain rats for a period of up to seven weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels 50, 150 and 500 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 500 and 150 mg/kg bw/day. These included reduced initial body weight gains in either sex at 500 mg/kg bw/day, reduced food consumption and increased water consumption in males at 500 mg/kg bw/day, organ weight changes in either sex at 500 and 150 mg/kg bw/day, macroscopic changes in males at 500 and 150 mg/kg bw/day and microscopic changes in either sex at 500 and 150 mg/kg bw/day. A No Observed Effect Level (NOEL) for systemic toxicity was considered to be 50 mg/kg bw/day for either sex.

The inflammatory cell infiltration in the periportal area of the liver in either sex at 500 mg/kg bw/day was considered to represent an adverse effect of treatment. The microscopic liver changes evident in females treated with 150 mg/kg bw/day were considered to be an adaptive response to mixed function oxidase induction (Capen C.C et al; 2002, Cattley R.C et al; 2002 and Zabka T.S et al; 2011). Therefore, a No Observed Adverse Effect Level (NOAEL) can be established at 150 mg/kg bw/day for females. Although the kidney findings of tubular basophilia and proteinaceous casts in male kidneys could be considered an adverse effect, these findings were considered to be associated with alpha 2u-globulin and formation of hyaline droplets, an effect recognized as being both species and sex specific and not relevant for humans. In terms of risk assessment, these findings observed on this study would suggest that a No Observed Adverse Effect Level (NOAEL) can be established at 150 mg/kg bw/day for males because the findings do not reflect true systemic toxicity.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Read across
System:
hepatobiliary
Organ:
liver

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 study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The repeated dose toxicity of the analogue substance, di-tert-butyl sec-butylidene diperoxide, was tested as a 50% solution in isododecane (Trigonox D-B50) in an OECD 422 study (Allt, 2017). Pure isododecane was also tested for repetad dose toxicity in an OECD 408 study (Suberg et al., 1983). Both Trigonox D-B50 and isododecane induced an increase of the liver weight associated with hepatocellular hypertrophy and male rat-specific alpha-2u globuline nephropathy. Therefore the toxicity of Trigonox D-B50 can be due, at least partially, to isododecane. Partially, because dialkyl organic peroxides, like di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide (CAS no. 78-63-7), di-tert-amyl peroxide (CAS no. 10508-09-5) and di-tert-butyl peroxide (CAS 110-05-4 ) tested as pure peroxides induced also a liver hypertrophy and male rat-specific nephropathy in repeated dose toxicity studies. Therefore, excluding the male rat-specific nephropathy, the NOAEL of 150 mg/kg/d of Trigonox DB-50 is equivalent to a NOAEL of 75 mg/kg/b for pure di-tert-butyl sec-butylidene diperoxide.

Additional information

Justification for classification or non-classification

No classification is warranted according to CLP and GHS criteria.