Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics, other
Type of information:
calculation (if not (Q)SAR)
Adequacy of study:
key study
Reliability:
other: A written assessment based on toxicological profile of the substance
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
A written assessment of toxicokinetic behaviour is considered appropriate for the substance. The substance displays only minor toxicological effects in any of the studies proposed, and is deemed to be be not harmful for health effects. As such, it is deemed inappropriate in terms of animal welfare to conduct a toxicokinetic assessment when no harmful effects are predicted based on known toxicology. A written assessment has therefore been prepared to address this endpoint.

Data source

Materials and methods

Objective of study:
other: Assessment of toxicokinetic behaviour
Principles of method if other than guideline:
Written assessment based on toxicological profile.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Not applicable

Test animals

Species:
other: Not applicable

Administration / exposure

Details on exposure:
Not applicable
Duration and frequency of treatment / exposure:
Not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
Not applicable
No. of animals per sex per dose:
Not applicable
Positive control:
Not applicable
Details on study design:
Not applicable
Details on dosing and sampling:
Not applicable
Statistics:
Not applicable

Results and discussion

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
Not applicable

Any other information on results incl. tables

Toxicokinetic parameters such as uptake, distribution, metabolism and excretion form the essential toxicological profile of a substance. An approximate indication of the toxicokinetic pattern can be gained from the physico-chemical properties (solubility in solvents, log Pow, hydrolytic stability) and the results of basic toxicity testing of the test article.

 

This substance has been supported under Environmental Protection Agency’s (EPA’s) High Production Volume (HPV) Challenge Program. The American Chemical Councils RAPA Panel, has derived a “Substituted Diphenylamines” category of chemicals for this substance, please refer to EPA reference 201-14700A located at

 

http://www.epa.gov/hpv/pubs/summaries/subdipha/c13378rt.pdf

 

Relying on several factors specified in EPA’s guidance document on “Development of Chemical Categories in the HPV Challenge Program,” in which use of chemical categories is encouraged, the chemicals constitute a chemical category on the following basis:

 

Structural Similarity. A key factor supporting the classification of these chemicals as a category is their structural similarity. All share a common starting material; Diphenylamine (Benzenamine, N-phenyl-, CAS# 122-39-4), a common synthetic pathway, and all compounds in this category are diamines with various substitutions.

 

Similarity of Physicochemical Properties. The similarity of the physicochemical properties of these materials parallels their structural similarity. All are off-white to light brown solids or viscous liquids intended for use as antioxidants in finished rubber articles or as antidegradant additives that extend the useful life of heavy-duty industrial functional fluids used in high-speed, high-temperature and/or high-load applications. As a class, these amine-based antidegradant compounds are less migratory (more polymer-bound) and less staining than the Substituted p-Phenylenediamine antidegradants. The use of these materials requires that they be stable under high temperatures. Their low volatility is due to their low vapor pressure, highly viscous or solid form. The existing information for these materials indicates that they have low water solubility and high flash points.

 

Toxicological Similarity. Review of existing published and unpublished test data for Substituted Diphenylamines shows the mammalian toxicity among the materials within this category are similar.

 

Conclusion. Based upon the data reviewed in “Substituted Diphenylamines” category of chemicals under the (EPA’s) High Production Volume (HPV) Challenge Program, the physicochemical and toxicological properties of the Substituted Diphenylamine category members are similar and follow a regular pattern as a result of that structural similarity. Therefore, the definition of a chemical category has been met, and read across is considered appropriate for the category of chemical.

 

The assessment of the toxicokinetic properties of the substance given below is therefore based on the results obtained for the substance and for analogues that are read across to. The following toxicological endpoints are utilised in this assessment:

 

·         Acute oral toxicity in rats (read across)

·         Acute dermal toxicity in rats (read across)

·         In vivo skin irritation in rabbits (read across)

·         Skin sensitization in guinea pigs (read across) and humans (substance itself)

·         Bacterial reverse mutation test

·         In vitro chromosome aberration test in human lymphocytes

·         In vivo micronucleus test in the mouse (read across)

·         Subacute (28-day) and subchronic oral toxicity in rats

·         Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity  Screening Test in the Rat. (read across)

 

The studies are sourced from the OECD SIDS data set utilised in the HPV programme, and are considered appropriate for use in the assessment of the substance.

 

Physico-chemical properties

The substance is an organic viscous UVCB liquid with a molecular weight range of 169.23 to 618.08 g/mol. It is characterized by a low water solubility of 0.706 mg/l at 20 °C and a relatively high partition coefficient (log Pow 5.2). 

Toxicological Profile:

The group was tested for acute oral toxicity in male and female rats on two separate analogues - Benzenamine, 2-ethyl-N-(2-ethylphenyl)-, (tripropenyl) derivatives (CAS 68608-77-5) and Benzenamine, N-phenyl-, styrenated (CAS 68442-68-2). Assessment of both results indicates that there is no hazard associated with short term toxicity to this group of substances; inferring that should adsorption occur following ingestion, the results are negligible. Read across is deemed appropriate to the substance subject to the registration, as the group demonstrates these effects as a whole.

 

Dermal treatment on two separate analogues - Benzenamine, 2-ethyl-N-(2-ethylphenyl)-, (tripropenyl) derivatives (CAS 68608-77-5) and Benzenamine, N-phenyl-, styrenated (CAS 68442-68-2) in the rabbit was also assessed. Skin reactions were limited to mild erythema, desquamation, and edema. Only barely perceptible to slight erythema and desquamation were present at day 14 of the studies. There is therefore evidence of absorption through the skin, although the results are again negligible. The substance is not deemed to be a skin sensitiser, on the basis of read across results and human patch testing data, inferring that the substituted diphenylamines do not elicit a sensitising response.

 

The substance was not mutagenic in two standard Ames Tests in Salmonella typhimurium (TA 98, TA 100, TA1535, TA1537) and E. coli WP2 uvr A. The substance was also non-clastogenic in an in vitro chromosome aberration test. Finally, the analogue Benzenamine, N-phenyl-, styrenated (CAS 68442-68-2) was negative in an in vivo micronucleus test in Mice. As such, it is considered inappropriate to conduct a further assessment ofin vitrogenotoxicity when available mutagenicity studies on the substance and other group members clearly confirm that the substance is not genotoxic.

 

There are two sets of data included for assessment of the repeated dose effects for the substance.  A feeding study on the substance itself followed a daily dietary administration for 64 weeks. This was found to retard growth in females at dose levels of 2500 ppm and higher. However, no effect on growth in males at 2500 ppm was noted. Liver enlargement was noted at all concentrations in both sexes along with diffuse hepatic degeneration. However, the severity of the liver changes were not considered to be treatment-related. The degenerative changes in the liver were described as diffuse cloudy swellings and fatty metamorphosis of the cytoplasm of the hepatocyte. No compound-related hematopoietic changes were observed in any of the test groups.  

 

An oral gavage study on the read across substance Benzenamine, N-phenyl-, styrenated (CAS 68442-68-2) administered at dose levels of 600, 250 and 50 mg/kg/day for 28-days resulted in treatment-related effects at all dose levels. Elevated alkaline phosphatase levels were detected for males treated with 600 mg/kg/day. Males treated with 600 and 250 mg/kg/day also showed reduced cholesterol levels. Elevated liver and adrenal weights, both absolute and relative to terminal bodyweights, were detected for animals of either sex treated with 600 mg/kg/day. Centrilobular hepatocyte enlargement was observed for animals of either sex treated with 600 and 250 mg/g/day, with the effect extending into the female 50 mg/kg/day dose group. Follicular cell hypertrophy was observed for males treated with 600 and probably also at 250 mg/kg/day, although the results at this level are not clear. The above results implies that a level of adsorption and subsequent metabolism is undertaken following ingestion; however this appears to be at higher dose levels. The effects noted are not considered to be reflective of true toxicity, and instead are adaptive responses. Therefore the ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 600 mg/kg/day, and this value is taken for the substance for hazard assessment on the basis of a “worst case” value.

The above oral gavage study on the read across substance Benzenamine, N-phenyl-, styrenated (CAS 68442-68-2) contained a reproduction / developmental toxicity screening element. No adverse effects on mating performance, fertility or gestation were detected. Litter Observations demonstrated no clinical signs to suggest an effect of treatment. Mean offspring weights for treated animals were comparable to controls. For developmental purposes, offspring from the 600 mg/kg/day dose group showed less successful completion of surface righting assessments. There were no treatment-related differences in pinna unfolding. No treatment-related macroscopic abnormalities were detected for the interim death offspring or for the remaining offspring at terminal kill. It was considered that treatment-related effects on reproduction were observed at 600 mg/kg/day. These were confined to an increase in pre-implantation losses, resulting in lower offspring numbers at this dose level. The NOAEL for reproductive toxicity was therefore considered to be 250 mg/kg/day.

 

Evaluation and Assessment

The substance is a substituted diphenylamine UVCB substance.   Substituted Diphenylamines, which are used as antidegradants in rubber, foamed polymers and high-temperature functional fluids (lubricants, gear oils, hydraulic fluids), are defined as amines with various substitutions. Their use in these applications requires stability at high temperatures, low biodegradation, low water solubility and low vapor pressure.Substituted Diphenylaminesas a group have been assessed under the US HPV programme in the “Substituted DiphenylaminesCategory”, sponsored by the American Chemistry Council’s Rubber and Plastic Additives Panel (RAPA). The findings appear to indicate that no significant mammalian toxicity is associated with this group.  This is supported by known data on the parent substance, diphenylamine.

 

Based on all available data, the substance therefore does not appear to contribute significantly to toxicokinetic behaviour.

 

The data of the acute dermal toxicity and dermal irritation test indicate limited dermal permeability, owing to the fact that only minor systemic and irritating effects were observed. However, these were observed to be almost fully reversible within the respective test periods, implying that any absorption could result in rapid metabolism or that the results observed could be due to the animals cleansing themselves rather than actual absorption taking place.

 

Taking the results of the sub-acute oral toxicity and developmental toxicity study into account, the substance appears to be absorbed from the gastrointestinal tract as evidenced by the liver and adrenal effects in the higher dose groups. This effect is anticipated, as it is known that the parent substance, diphenylamine is well absorbed from the gastrointestinal tract in man and in several animal species including rat, rabbit, dog and cow. Results for diphenylamine indicate that up to 3 % of the parent compound and approximately 80-90 % of the dose is excreted as 12 different metabolites, which include 4-hydroxydiphenylamine, 4,4’- 2 hydroxydiphenylamine and sulfate and glucuronide conjugates of these hydroxylated metabolites. In addition, indophenol has been identified as metabolite. From these results it can be assumed that a similar mechanism to that employed for diphenylamine is utilised in elimination processes for the substituted diphenylamine group. The substance is proposed to be readily metabolized and excreted and that accumulation seems to be unlikely. There are no data on dermal route of administration or exposure by inhalation. An absorption rate of 100% for the oral route is proposed to be taken for risk characterisation purposes, whereas dermal and inhalation absorption is assumed to be 100% (defaults). The assumption of a default dermal absorption value of 100% is supported by the effects noted in the irritation studies. Due to the potential for absorption and the lack of experimental data, a default absorption value of 100% is also assumed for inhalative uptake.

 

As all other effects noted are presumed to be adaptive, the substance is proposed not to have significant effects following adsorption. 

The substance has been demonstrated to have a partition coefficient value which indicates the potential to accumulate biologically. However evaluation using recognised QSAR programmes indicates that the substance is unlikely to bioaccumulate in body tissues. As a UVCB substance, there are a wide range of composite substances that make up the product. A sample of potential substances has been assessed, and the overall conclusion was that these substances are unlikely to bioaccumulate. This is further emphasised by the bioaccumulation potential of the parent substance, Diphenylamine which demonstrates only a low level bioconcentration factor in fish (BCFfish of 155). In conclusion, the highly variable structures of the substance is proposed not to fall into a BCF level where classification is applicable. These factors indicate that accumulation and distribution within body tissues is unlikely to occur. 

Conclusion

The results of basic toxicity testing give no reason to anticipate unusual characteristics with regards to the toxicokinetics of the substance. The data indicate that whilst there is the possibility of potential dermal absorption, there are no predicted effects noted from this route. Minor systemic effects associated with absorption potential have been observed following oral ingestion; however, these appear to be adaptive and are negated, following cessation. Bioaccumulation and storage of the material in fatty tissue (adipose cells) of the substance can most probably be excluded due to the predictive assessment of bioaccumulation behaviour using QSAR tools and the known experimental values of the parent substance, diphenylamine. Based on the results of all mutagenicity assays and assessment of relevant literature, a metabolisation towards genotoxic structures can be ruled out. It is proposed that following ingestion, the substance will be hydrolysed in the stomach to more soluble forms, followed by further metabolism via oxidation using standard metabolic mechanisms to carbon dioxide and soluble forms for subsequent elimination. 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
The results of basic toxicity testing give no reason to anticipate unusual characteristics with regards to the toxicokinetics of the substance. The data indicate that whilst there is the possibility of potential dermal absorption, there are no predicted effects noted from this route. Minor systemic effects associated with absorption potential have been observed following oral ingestion; however, these appear to be adaptive and are negated, following cessation. Bioaccumulation and storage of the material in fatty tissue (adipose cells) of the substance can most probably be excluded due to the predictive assessment of bioaccumulation behaviour using QSAR tools and the known experimental values of the parent substance, diphenylamine. Based on the results of all mutagenicity assays and assessment of relevant literature, a metabolisation towards genotoxic structures can be ruled out. It is proposed that following ingestion, the substance will be hydrolysed in the stomach to more soluble forms, followed by further metabolism via oxidation using standard metabolic mechanisms to carbon dioxide and soluble forms for subsequent elimination.
Executive summary:

The substance is a substituted diphenylamine UVCB substance.   Substituted Diphenylamines, which are used as antidegradants in rubber, foamed polymers and high-temperature functional fluids (lubricants, gear oils, hydraulic fluids), are defined as amines with various substitutions. Their use in these applications requires stability at high temperatures, low biodegradation, low water solubility and low vapor pressure.Substituted Diphenylamines as a group have been assessed under the US HPV programme in the “Substituted Diphenylamines Category”, sponsored by the American Chemistry Council’s Rubber and Plastic Additives Panel (RAPA). The findings appear to indicate that no significant mammalian toxicity is associated with this group.  This is supported by known data on the parent substance, diphenylamine.

 

Based on all available data, the substance therefore does not appear to contribute significantly to toxicokinetic behaviour.

 

The data of the acute dermal toxicity and dermal irritation test indicate limited dermal permeability, owing to the fact that only minor systemic and irritating effects were observed. However, these were observed to be almost fully reversible within the respective test periods, implying that any absorption could result in rapid metabolism or that the results observed could be due to the animals cleansing themselves rather than actual absorption taking place.

 

Taking the results of the sub-acute oral toxicity and developmental toxicity study into account, the substance appears to be absorbed from the gastrointestinal tract as evidenced by the liver and adrenal effects in the higher dose groups. This effect is anticipated, as it is known that the parent substance, diphenylamine is well absorbed from the gastrointestinal tract in man and in several animal species including rat, rabbit, dog and cow. Results for diphenylamine indicate that up to 3 % of the parent compound and approximately 80-90 % of the dose is excreted as 12 different metabolites, which include 4-hydroxydiphenylamine, 4,4’- 2 hydroxydiphenylamine and sulfate and glucuronide conjugates of these hydroxylated metabolites. In addition, indophenol has been identified as metabolite. From these results it can be assumed that a similar mechanism to that employed for diphenylamine is utilised in elimination processes for the substituted diphenylamine group. The substance is proposed to be readily metabolized and excreted and that accumulation seems to be unlikely. There are no data on dermal route of administration or exposure by inhalation. An absorption rate of 100% for the oral route is proposed to be taken for risk characterisation purposes, whereas dermal and inhalation absorption is assumed to be 100% (defaults). The assumption of a default dermal absorption value of 100% is supported by the effects noted in the irritation studies. Due to the potential for absorption and the lack of experimental data, a default absorption value of 100% is also assumed for inhalative uptake.

 

As all other effects noted are presumed to be adaptive, the substance is proposed not to have significant effects following adsorption. 

The substance has been demonstrated to have a partition coefficient value which indicates the potential to accumulate biologically. However evaluation using recognised QSAR programmes indicates that the substance is unlikely to bioaccumulate in body tissues. As a UVCB substance, there are a wide range of composite substances that make up the product. A sample of potential substances has been assessed, and the overall conclusion was that these substances are unlikely to bioaccumulate. This is further emphasised by the bioaccumulation potential of the parent substance, Diphenylamine which demonstrates only a low level bioconcentration factor in fish (BCF fish of 155). In conclusion, the highly variable structures of the substance is proposed not to fall into a BCF level where classification is applicable. These factors indicate that accumulation and distribution within body tissues is unlikely to occur. 

Conclusion

The results of basic toxicity testing give no reason to anticipate unusual characteristics with regards to the toxicokinetics of the substance. The data indicate that whilst there is the possibility of potential dermal absorption, there are no predicted effects noted from this route. Minor systemic effects associated with absorption potential have been observed following oral ingestion; however, these appear to be adaptive and are negated, following cessation. Bioaccumulation and storage of the material in fatty tissue (adipose cells) of the substance can most probably be excluded due to the predictive assessment of bioaccumulation behaviour using QSAR tools and the known experimental values of the parent substance, diphenylamine. Based on the results of all mutagenicity assays and assessment of relevant literature, a metabolisation towards genotoxic structures can be ruled out. It is proposed that following ingestion, the substance will be hydrolysed in the stomach to more soluble forms, followed by further metabolism via oxidation using standard metabolic mechanisms to carbon dioxide and soluble forms for subsequent elimination.