Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics, other

Type of information:

other: Review based on results of toxicological studies

Adequacy of study:

key study

Study period:

28 April 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Data source

Reference

Reference Type:

other: Review of study data

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Objective of study:

toxicokinetics

Principles of method if other than guideline:

Review of the study data therefore no guideline was followed.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Formaldehyde, oligomeric reaction products with acetone and diphenylamine

EC Number:

500-011-5

EC Name:

Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Cas Number:

9003-80-9

Molecular formula:

UVCB substance - not applicable

IUPAC Name:

N-phenylaniline; formaldehyde; propan-2-one

Test material form:

solid: flakes

Details on test material:

Identification: Formaldehyde, oligomeric reaction products with acetone and diphenylamine
Appearance: Dark brown flakes
Batch: IC5B04P006
Purity/Composition: 100% Unknown or Variable compositions, Complex reaction products and Biological materials (UVCB)
Test substance storage: At room temperature
Stable under storage conditions until: 26 February 2019 (expiry date)
Purity/composition correction factor: No correction factor required
Test substance handling: No specific handling conditions required
CAS Number: 9003-80-9
Molecular structure: UVCB
Molecular formula: UVCB
Molecular weight: UVCB

Results and discussion

Any other information on results incl. tables

Introduction

Assessment of the toxicokinetic behaviour of a substance includes evaluation of the absorption, distribution, metabolism and excretion (ADME) of the substance. ADME describes the uptake of a substance into the body and its lifecycle within the body, (including excretion) and can be defined as follows:

•            ABSORPTION: how, how much, and how quickly the substance enters the body;

•            DISTRIBUTION: reversible transfer of substances between various parts of the organism, i.e. body fluids or tissues;

•            METABOLISM: the enzymatic or non-enzymatic transformation of the substance of interest into a structurally different chemical (metabolite);

•            EXCRETION: the physical loss of the parent substance and/or its metabolite(s); the principal routes of excretion are via the urine, bile (faeces), and exhaled air.

•            Metabolism and excretion are the two components of ELIMINATION, which describe the loss of substance by the organism, either by physical loss or by chemical transformation.

The sum of processes following absorption of a chemical into the circulatory systems, distribution throughout the body, biotransformation, and excretion is called DISPOSITION.

 

A written assessment of toxicokinetic behaviour is considered appropriate for the substance, formaldehyde, oligomeric reaction products with acetone and diphenylamine.

 

Data set

The following data set is available for the substance:

Chemical name (UVCB): Formaldehyde, oligomeric reaction products with acetone and diphenylamine

EC No. / CAS No.: 500-011-5 / 9003-80-9

Molecular weight range for components: 165 to 569.28

State of the substance at 20°C and 101.3kPa: Dark, brown flakes

Vapour pressure: 0.095 Pa at 20 °C

Solubility: 0.276 mg/l, 0.401 mg/l and 1.48 mg/l at a flow rate of 24 ml/h, 12 ml/h and 6 ml/h respectively.

Partition coefficient n-octanol/water: Log Kow > 6.5

In vivo skin irritation: Not classified as an irritant

In vivo eye irritation: Not classified as an irritant

Skin sensitisation: Sensitising in mouse local lymph node assay.

in vitro mutagenicity (Ames study, in vitro transformation assay, in vitro mammalian gene mutation study, in vitro mouse lymphoma forward mutation assay, in vitro chromosome aberration test): Not classified as mutagenic

Acute toxicity - oral route: LD50 > 5000 mg/kg

Acute toxicity -  dermal route: LD50 > 2000 mg/kg

Acute toxicity – inhalation route: LC50 (4 hour) > 5 mg/L

28-day combined repeated dose toxicity study with the reproduction / developmental toxicity screening test: Parental NOAEL: 50 mg/kg; Reproduction NOAEL: at least 500 mg/kg.; Developmental NOAEL: 50 mg/kg. Classified as STOT RE 2, H372.

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 results of basic toxicity testing. The assessment of the toxicokinetic properties of formaldehyde, oligomeric reaction products with acetone and diphenylamine detailed below is based on the results obtained for the above physical/chemistry and toxicological endpoints. 

 

Overview of toxicological profile:

In the key oral toxicity study, a single oral dose of 5000 mg/kg of the test material was administered by oral gavage to two subsequent groups of three female Wistar rats. No mortality occurred. Hunched posture, piloerection and/or uncoordinated movements were noted for all animals on Day 1. The body weight gain shown by the animals over the study period was considered to be similar to that expected for normal untreated animals of the same age and strain. Therefore, the acute oral median lethal dose (LD50) was determined to be > 2000 mg/kg body weight. According to the OECD 423 test guideline, the LD50 cut-off value was considered to exceed 5000 mg/kg body weight.

In an acute dermal toxicity study, the test material was administered to five Wistar rats of each sex by a single dermal application at 2000 mg/kg body weight for 24 hours. No mortality occurred. Flat posture, ptosis and/or chromodacryorrhoea (snout) were noted for all animals on Day 1. Black discoloration was seen in the treated skin-area of all animals on Days 2 and/or 3. This was considered due to the colour of the formulation. Scales and/or scabs were noted for animals no. 3, 4, 5 and 9 between Days 5 and 15. These local effects were considered not to have affected the conclusion of the study. The mean body weight gain during the observation period was within the range expected for rats used in this type of study. No abnormalities were found at macroscopic post mortem examination of the animals. Therefore, the acute dermal median lethal dose (LD50) was determined to be > 2000 mg/kg body weight.

In an acute inhalation toxicity study, doses of 1 mg/L and of 5 mg/L of the test material were administered as dust by nose-only inhalations for 4 hours to two groups of five male and five female Wistar rats, respectively. No mortality occurred. At 1 mg/L, quick breathing was seen during exposure. After exposure, one male showed hunched posture on Day 1. Brown staining by test item remnants of the skin and fur was seen between Days 1 and 6. At 5 mg/L, quick breathing was seen during exposure. After exposure, no systemic clinical signs were seen. Brown and green staining by test item remnants of the skin and fur was seen between Days 1 and 4. Overall body weight gain in males and females was within the range expected for rats of this strain and age used in this type of study and were therefore considered not indicative of toxicity. Macroscopic post mortem examination revealed several purple foci on the left side of the thymus of one female exposed to 1 mg/L. Macroscopic examination of the other animals did not reveal further abnormalities. Therefore, the acute inhalation median lethal concentration (LC50) was determined to be > 5 mg/L

In the in vivo skin irritation study, 0.5 g of the test material moistened with 50% watery ethanol was applied onto clipped skin of rabbits for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours after exposure. No skin irritation occurred 4 hours after exposure to formaldehyde, oligomeric reaction products with acetone and diphenylamine. The results of an in vitro skin irritation test and an in vitro skin corrosion test also showed that the test material is not a skin irritant or corrosive to the skin, respectively. Therefore, the substance is not classified as a skin irritant in accordance with the CLP Regulation.

In the in vivo eye irritation study, single samples of approximately 70.8 mg of Formaldehyde, oligomeric reaction products with acetone and diphenylamine (a volume of approximately 0.1 mL) were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours after instillation. Irritation of the conjunctivae, which consisted of redness, chemosis and discharge, was observed. The irritation had completely resolved within 48 hours in one animal and within 72 hours in the other two animals. In addition, no eye irritation was observed in an in vitro eye irritation test conducted with the substance. Therefore, these results do not trigger classification of the substance as an eye irritant in accordance with the CLP Regulation.

In a local lymph node assay in the mouse, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)). Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1562, 2207 and 5300 DPM, respectively. The mean DPM/animal value for the vehicle control group was 487 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 3.2, 4.5 and 10.9, respectively. These results show that the test substance elicits a SI ≥ 3. The data showed a dose-response and the EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between >0 and 10%. The results of this study trigger classification of the substance as a skin sensitiser (Skin Sens. 1) in accordance with the CLP Regulation.

An oral combined repeated dose toxicity study with the reproduction/developmental toxicity screening study (in accordance with OECD Guideline 422) was conducted with the substance. The substance was administered by gavage to three groups, each of 10 male and 10 female SPF-bred Wistar Han rats, at dose levels of 50, 150 and 500 mg/kg body weight/day. A control group of 10 males and 10 females was dosed with vehicle alone (polyethylene glycol 400). Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females that delivered were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13 days of lactation (mostly for 50-56 days). Females that failed to deliver offspring were treated for 41 days.

Body weight gain was decreased, to about the same extent, in females at 150 and 500 mg/kg throughout the lactation period. This was accompanied by decreased body weights at 500 mg/kg (up to 10% lower than controls at Day 13 of lactation) and decreased food consumption between lactation Days 7-13 at 150 and 500 mg/kg. Food consumption during gestation, on the other hand, was increased in females at 500 mg/kg and, to a lesser extent, 150 mg/kg. Adverse, treatment-related microscopic changes were observed in the liver starting at 150 mg/kg, generally in both sexes. These changes were characterized by single cell necrosis (up to moderate degree), increased mitosis (up to slight degree), pigmentation of Kupffer cells/macrophages (up to slight degree; not observed in 150 mg/kg males) and hypertrophy/hyperplasia of Kupffer cells (females only). Liver weight was slightly decreased in females at 500 mg/kg but this could not clearly be related to the microscopic liver findings. At 500 mg/kg (in both sexes), the histopathological changes in the liver were accompanied by increases in the plasma levels of alanine and aspartate aminotransferase (ALAT, ASAT) alkaline phosphatase (ALP), bilirubin and bile acids, and decreases in total protein and albumin. The increased ASAT and ALP values in a few females at 50 mg/kg were not corroborated by histopathological changes and therefore considered to be non-adverse. Prothrombin time (PT) was increased in males at 500 mg/kg. There was no evidence of impaired coagulation.

No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg). At postnatal day 13, body weights of male and female pups were decreased at 150 and 500 mg/kg (by about 10 and 15%, respectively). Pup body weights at postnatal day 1 were similar across the groups, indicating that the lower weights at postnatal day 13 resulted from reduced post-natal growth. The serum level of thyroid hormone T4 was decreased in male and female pups at 500 mg/kg (on average by about 25-30%). These developmental effects were considered to be toxicologically relevant. They occurred in the presence of maternal toxicity. No developmental toxicity was observed in the absence of maternal toxicity. Based on these results, the following No Observed Adverse Effect Levels (NOAELs) were derived: parental NOAEL of 50 mg/kg, reproduction NOAEL of at least 500 mg/kg and Developmental NOAEL of 50 mg/kg (based on reduced post-natal growth starting at 150 mg/kg, accompanied by a decrease in serum T4 at 500 mg/kg).

 

The potential mutagenicity of the substance was evaluated in the following four in vitro mutagenicity studies:

•            In an Ames study, the results for test material were negative in strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 of Salmonella typhimurium both with and without metabolic activation preparation at 1000, 333, 100, 33 and 10 μg/plate.

•            In an in vitro transformation assay, the test material did not induce the appearance of a significant number of transformed foci over the applied concentration range of 10.0 μg/ml to 1000.0 μg/ml. This concentration range encompassed the range of apparent solubilities of the test material and corresponded to approximately 80% to 67% survival in the cytotoxicity test. Therefore, the test material is considered to be inactive in the BALB/3T3 in vitro transformation assay.

•            In an in vitro mouse lymphoma forward mutation assay, the test material induced small increases in the mutant frequency of the TK locus in L5178Y mouse lymphoma cells only in the presence of rat liver S-9 microsomal activation. Without activation, concentrations up to 1000 μg/ml were moderately toxic and were not detectably mutagenic. With activation, only highly toxic treatments at concentrations at or above 800 μg/ml were weakly mutagenic in the second trial. In the third trial, concentrations of test material from 400 μg/ml to 1000 μg/ml were moderately toxic and induced weak but significant increases in the mutant frequency. The results from the first trial were not discussed because of technical problems with the assay. Therefore, the test material is considered to be weakly active with metabolic activation.

•            In an in vitro chromosome aberration test, the substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No biologically relevant effects of Formaldehyde, oligomeric reaction products with acetone and diphenylamine on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Based on the results of the available in vitro studies, the substance is not classified as mutagenic.

 

 

Toxicokinetic parameters:

 

Absorption

 

Oral

No mortality was observed in the acute oral toxicity study in rats at a dose level of 2000 mg/kg body weight, however, signs of toxicity were observed in rats on day 1. In the oral combined repeated dose toxicity study with the reproduction/developmental toxicity screening study in rats, adverse microscopic alterations in the liver and associated changes in clinical pathology parameters were observed in both sexes, and reduced body weight gain and food consumption during lactation occurred in females at the higher dose levels. Therefore, the results of these oral toxicity studies indicate that the substance is absorbed by the oral route.

 

The molecular weight range of the components of this substance is 165 to 569.28; molecular weights below 500 are favourable for absorption therefore components with molecular weights below 500 may be absorbed. The substance is poorly soluble in water and water-insoluble substances will not readily dissolve into the gastrointestinal fluids. Log Kow values between -1 and 4 are favourable for absorption by passive diffusion; the substance has an estimated log Kow value of > 6.5, which does not favour absorption by passive diffusion.

 

As a worst case, an oral absorption rate of 100% will be used for the chemical safety assessment.

 

 

Dermal

In the acute dermal toxicity study in rats at a dose level of 2000 mg/kg, no mortality occurred, however, flat posture, ptosis and/or chromodacryorrhoea (snout) were noted for all animals on Day 1. 

In the in vivo skin irritation study, no skin irritation was observed after 4 hours of exposure to the substance. In a mouse local lymph node assay, the substance was shown to be a skin sensitiser, indicating that some uptake of at least the lower molecular components occurs by the dermal route, although it may only be a very small fraction of the applied dose.

The molecular weight range of the components of this substance is 165 to 569.28; a molecular weight of less than 100 favours dermal uptake, however, molecules with molecular weight above 500 may be too large. The log Kow value for the substance is estimated to be > 6.5 therefore the dermal absorption of the substance is predicted to be limited based on the high log Kow value. Log Kow values between 1 and 4 favour dermal absorption (values between 2 and 3 are optimal) particularly if water solubility is high. At log Kow value above 6, the rate of transfer between the stratum corneum and the epidermis will be slow and will limit absorption across the skin; uptake into the stratum corneum itself may also be slow. The substance is also poorly soluble in water, which does not favour dermal absorption; the substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis.

 

In accordance with ECHA Chapter R.7c: Endpoint specific guidance, a default value of 100% skin absorption is generally used unless the molecular weight is above 500 and the log P/Kow is outside the range [-1, 4], in which case a value of 10% skin absorption is chosen. Several of the substance have a molecular weight of < 500 therefore a dermal absorption rate of 100% will be used for the chemical safety assessment, as a worst case.

 

Inhalation

No mortality was observed in an acute inhalation toxicity study at dose levels up to 5 mg/L. At 1 mg/L, one male showed hunched posture on Day 1 after exposure. Macroscopic post mortem examination revealed several purple foci on the left side of the thymus of one female exposed to 1 mg/L. Macroscopic examination of the other animals did not reveal further abnormalities.

The substance has an estimated log Kow value of > 6.5; moderate log Kow values (between -1 and 4) are favourable for absorption directly across the respiratory tract epithelium by passive diffusion. Consequently, poor absorption of the substance by inhalation may be predicted.

 

As a worst case, an oral absorption rate of 100% will be used for the chemical safety assessment for this substance.

 

Distribution

There are no data available on distribution of the substance. Adverse effects on the liver were observed in the oral, repeated dose study, indicating that components of the substance are distributed to the liver.

 

Metabolism and excretion

The components of the substance, which are absorbed, are likely to undergo metabolism to more polar conjugated metabolites, which are then excreted. The results of the in vitro mutagenicity studies indicate that the substance is not metabolised to genotoxic structures.

 

Bioaccumulation of the substance may be expected based on its estimated high log Kow value and low water solubility. However, the substance is not predicted to be bioaccumulative based on the results of an evaluation of the main components of the substance using available QSAR models.

 

Conclusion

The results of these oral toxicity studies indicate that the substance is absorbed by the oral route. Molecular weights below 500 are favourable for absorption therefore components of the substance with molecular weights below 500 may be absorbed. The substance is poorly soluble in water and water-insoluble substances will not readily dissolve into the gastrointestinal fluids. The substance has an estimated log Kow value of > 6.5, which does not favour absorption by passive diffusion.

 

In accordance with ECHA Chapter R.7c: Endpoint specific guidance, a default value of 100% skin absorption is generally used unless the molecular weight is above 500 and the log P/Kow is outside the range [-1, 4], in which case a value of 10% skin absorption is chosen. Several of the components of the substance have a molecular weight of < 500 therefore a dermal absorption rate of 100% will be used for the chemical safety assessment, as a worst case.

 

The substance has an estimated log Kow value of > 6.5; moderate log Kow values (between -1 and 4) are favourable for absorption directly across the respiratory tract epithelium by passive diffusion. Consequently, poor absorption of the substance by inhalation may be predicted.

 

The following absorption factors will be used for the chemical safety assessment:

•            Dermal route: 100% (worst case)

•            Oral route: 100% (worst case)

•            Inhalation route: 100% (worst case)

 

There are no data available on distribution of the substance. Adverse effects on the liver were observed in the oral, repeated dose study, indicating that components of the substance are distributed to the liver. The substance is predicted not to be bioaccumulative.

 

References

 

ECETOC (European Centre for Ecotoxicology and Toxicology of Chemicals). Monograph No, 20; Percutaneous absorption. August 1993

 

ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7c: Endpoint specific guidance, Version 2.0, November 2014

 

Applicant's summary and conclusion

Conclusions:

The following default absorption factors will be used for the chemical safety assessment:
•            Dermal route: 100% (worst case)
•            Oral route: 100% (worst case) (50% if oral-to-inhalation extrapolation according to ECHA Guidance)
•            Inhalation route: 100% (worst case)
 
There are no data available on distribution of the substance. Adverse effects on the liver were observed in the oral, repeated dose study, indicating that components of the substance are distributed to the liver. The substance is predicted not to be bioaccumulative.

Executive summary:

The results of these oral toxicity studies indicate that the substance is absorbed by the oral route. Molecular weights below 500 are favourable for absorption therefore components of the substance with molecular weights below 500 may be absorbed. The substance is poorly soluble in water and water-insoluble substances will not readily dissolve into the gastrointestinal fluids. The substance has an estimated log Kow value of > 6.5, which does not favour absorption by passive diffusion.

 

In accordance with ECHA Chapter R.7c: Endpoint specific guidance, a default value of 100% skin absorption is generally used unless the molecular weight is above 500 and the log P/Kow is outside the range [-1, 4], in which case a value of 10% skin absorption is chosen. Several of the components of the substance have a molecular weight of < 500 therefore a dermal absorption rate of 100% will be used for the chemical safety assessment, as a worst case.

 

The substance has an estimated log Kow value of > 6.5; moderate log Kow values (between -1 and 4) are favourable for absorption directly across the respiratory tract epithelium by passive diffusion. Consequently, poor absorption of the substance by inhalation may be predicted.

 

The following absorption factors will be used for the chemical safety assessment:

•            Dermal route: 100% (worst case)

•            Oral route: 100% (worst case) (50% if oral-to-inhalation extrapolation according to ECHA Guidance)

•            Inhalation route: 100% (worst case)

 

There are no data available on distribution of the substance. Adverse effects on the liver were observed in the oral, repeated dose study, indicating that components of the substance are distributed to the liver. The substance is predicted not to be bioaccumulative.