Reaction mass of allyl (2-methylbutoxy)acetate and allyl (3-methylbutoxy)acetate

Endpoint summary

Description of key information

A toxicokinetic assessment was conducted in accordance with REACH Annex VIII 8.8.1. The substance Allyl Amyl Glycolate (AAG; EC No. 916-328-0) is a multi-constituent organic liquid. It has 2 main constituents: allyl (3-methylbutoxy) acetate (CAS No. 67634-00-8) which has a typical concentration of 79 % (71 – 85 % concentration range) and allyl (2-methylbutoxy) acetate (CAS No. 67634-01-9) which has a typical concentration of 20% (15 – 29 % concentration range). Several unknown impurities are present at a concentration below 1%.

A full ADME toxicokinetic study in the rat is not available. One in vivo study with AAG in rats covering the oral route is available (acute oral toxicity study). One in vivo study with allyl (3-methylbutoxy) acetate in the guinea pig covering the dermal route is available (guinea pig maximisation test). An oral combined repeated dose toxicity study with reproduction/developmental toxicity screening test with AAG in rats is available. No inhalational toxicity study data is available. Further details on endpoints are available in the IUCLID 6 registration dossier. The toxicokinetic analysis is based on the physicochemical and in vivo toxicological data.

The absorption rates of 50% (oral), 50% (dermal) and 100% (inhalation) are accepted for chemical risk assessment purposes.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

50

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

100

Additional information

1.Physicochemical properties

In accordance with the ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7C Section R.7.12 (Endpoint Specific Guidance), the physicochemical properties can provide an insight into the potential behaviour of Allyl Amyl Glycolate in the body.

Absorption - oral

The molecular weight of Allyl Amyl Glycolate is 186.25 g/mol and is in the range for favourable oral absorption (<500 g/mol). The log Pow (2.57 at 25°C) indicates it is moderately lipophilic and the water solubility (896.349 mg/L at 20°C) indicates it is moderately soluble in water. These characteristics will facilitate transport of Allyl Amyl Glycolate via passive diffusion.

Absorption – dermal

The physical state, log Kow and water solubility of Allyl Amyl Glycolate are in the optimal range for dermal absorption.

Absorption – inhalation

Allyl Amyl Glycolate has a moderate vapour pressure (0.42 kPa at 20˚C) and a boiling point of 224˚C. The substance is used in many consumer products such as cleaning agents and cosmetics; the main route of exposure is dermal, so inhalation exposure was not considered.

Distribution/Metabolism/Excretion

Based on the molecular weight, water solubility and log Kow, Allyl Amyl Glycolate is likely to be widely distributed and will be excreted as metabolites in the urine.

2. Information from other studies in the dossier

Absorption - oral

In an acute oral toxicity study (OECD 423/GLP) in female Wistar female rats, the LD50 (female) was >300 < 2000 mg/kg bw.

In a combined repeated dose and reproduction/developmental toxicity screening test (OECD 422/GLP), Allyl Amyl Glycolate was administered to Wistar rats (6 animals/sex/group for repeated dose toxicity; 12 animals/sex/group for reproduction/developmental toxicity screening) by gavage in olive oil at dose levels of 0, 30, 60 and 120 mg/kg bw/day (main study; 0 and 120 mg/kg bw/day for satellite animals), 7 days per week, for 49 days (parental males and all satellite animals) and until the end of the lactation period (parental females). Satellite animals were used for observation of reversibility, persistence or delayed occurrence of systemic toxicity effects for 14 days post treatment.

Repeated dose toxicity results

There were no unscheduled male or female deaths during the repeated dose toxicity part of the study. Body weight of females was significantly decreased during the lactation period at the dose level 120 mg/kg bw/day. Statistically significant differences in necropsy body weight were not found in satellite treated females. No differences in functional observations were noted in any males/females throughout the study.

Haematological examination of males at the dose level 120 mg/kg bw/day revealed statistically significantly changed parameters (erythrocyte count, haemoglobin concentration, MCV, reticulocytes, granulocytes, monocytes) in comparison with the control males were recorded. In satellite treated males, the value of MCV remained statistically significantly increased in comparison with the control satellite animals. Haematological examination of females demonstrated statistically significantly changed parameters (erythrocyte, haemoglobin concentration, haematocrite, total leucocyte count, APTT and PT) only in females at the dose level 120 mg/kg bw/day in comparison with the control females. In satellite treated females, some of the parameters were changed statistically insignificantly, but parameters were outside of the historical control range in comparison with the control satellite animals. During biochemical examination of males, statistically significantly changed values (compared to control) in liver enzyme concentrations were recorded.  In males at the dose level 120 mg/kg bw/day, statistically significantly increased concentrations of liver enzymes – ALP, ALT, AST. In males at 60 mg/kg bw/day, the value of ALP and potassium ions was also statistically significantly increased. In males at 30 mg/kg, the values of ALB and GLU were increased statistically significantly. In satellite treated males, an increased value of creatinine was recorded. During the biochemical examination of females, statistically significantly changed values (compared to control) in liver enzyme concentration were recorded.  In females at the dose level 120 mg/kg bw/day, there was an increase in ALP and T-Bil. Statistically significant decreases in Crea, BUN (out of historical control range) and GLU were recorded. In satellite treated females, significant decrease in the values of Ca and T-Bil was recorded. During urinalysis, the presence of proteins, blood and leucocytes were recorded in treated males as well as in control males and were not associated with the application of the test item.

In males at 120 mg/kg bw/day, the relative weight of spleen was increased statistically significantly. Absolute and relative weights of the kidneys and liver were statistically significantly increased. In males at the dose level 30 mg/kg bw/day, the absolute and relative weight of spleen was significantly decreased. In satellite treated males, a statistically significant increase in the absolute and relative weight of the prostate gland with seminal vesicles was recorded. In females at the dose level 120 mg/kg bw/day, a statistically significant increase in the absolute and relative weight of the spleen and a decrease in the absolute weight of the brain was recorded. Statistically significant differences were not recorded in the absolute or relative weights of organs in satellite females. Macroscopical findings in the liver and spleen were diagnosed in animals mainly at the dose level 120 mg/kg bw/day during the pathological examination. Multiple focal changes of light colour on the liver, altered liver appearance, enlarged spleen were recorded in males and females. Sporadic findings were diagnosed in animals at the dose level 60 mg/kg bw/day. No macroscopical findings were recorded in animals at the dose level 30 mg/kg bw/day. Histological examination recorded changes related to the test item treatment in males and females. Focal necrosis of differing severity and mostly of periportal localization were found in the liver parenchyma of 4/6 males and 4/6 females from the high dose group. Further, minimal to mild bile duct hyperplasia was observed in the liver of all 6 males and 6 females. The presence of small amounts of brown pigment (possibly hemosiderin) was noted in the liver of 1/6 males and 4/6 females. These lesions were in direct relation to the test item administered.  Recovered high dose rats showed minimal bile duct hyperplasia in the liver of 4/6 males and 2/6 females, together with the presence of a small amount of brown pigment in 2/6 and 2/6 females and a small solitary granuloma in 1/6 male and 2/6 females. Minimal periportal fibrosis was seen in the liver of 2/6 recovered males. Bile duct proliferation participates in the repair of liver damage. The test item has a direct hepatotoxic effect (liver necrosis), so the liver is a target organ. Bile duct proliferation observed is considered to be a part of reparative process, so its relation to the test item is most probably indirect. Mild to marked extramedullary hemopoiesis was found in the spleen of 3/6 males and 3/6 females from the high dose group and one recovered high dose female in comparison to its minimal grade found in one control male. This finding was most probably related to the test item administration and led to subsequent examination of the spleen in the middle and low dose groups of rats. Extramedullary hemopoiesis (mild grade) was found in the spleen of one middle dose male and of minimal grade in one low dose female.

The NOAEL (No Observed Adverse Effect Level) for repeated dose toxcity in males and females was established as 60 mg/kg body weight/day. This judgement is based predominantly on changes of biochemical parameters (liver enzymes), macroscopical and microscopical findings on liver and spleen in animals at the dose level 120 mg/kg bw/day. The NOEL (No Observed Effect Level) for repeated dose toxcity in males and females was established as 30 mg/kg body weight/day.

Reproduction/developmental toxicity screening test results

There were no male parental mortalities. One female was found dead on the 12th day of lactation (the reason of death was not detected, because of partial autolysis of organs). Statistically significant differences in necropsy body weight were not found in treated males. A statistically significant decrease in body weight was recorded in all treated groups of females on day 20 of pregnancy. Mean body weight of all treated groups was decreased (on day 4 and 12 of lactation statistically significantly, with the dose dependency) in comparison with controls.

The relative weight of reproductive organs (testes, epididymis and prostate gland with seminal) and pituitary gland of treated groups of males was comparable to the control males. The relative weight of the thyroid gland was statistically significantly increased in males at the dose level 120 mg/kg bw/day. The relative weight of reproductive organs (ovaries) were decreased in weight compared to the control females but it was not statistically significant. The absolute and relative weight of the thyroid gland of treated females was comparable to the control females. A comparison of sperm motility in the control males and males from treated groups did not show differences. The test item treatment did not affect sperm morphology. Male rat ability to produce sperm that can fertilise eggs was not affected by the test item administration.

No treatment-related macroscopical changes in the reproductive organs, pituitary and thyroid glands were noted in treated males or females. Microscopical examination of reproductive organs and the pituitary gland did not reveal presence of treatment-related changes. Toxicity of the test item was confirmed by the histological findings in the liver in the 12 males and 12 females. (Focal necrosis of the liver in 6/12 males and females and bile duct hyperplasia in 12/12 males and 9/12 females).

A decreased number of females achieving pregnancy was recorded at the dose level 60 and 120 mg/kg bw/day.  No abortions were recorded. The mean duration of pregnancy was similar in treated and control groups.  The mean number of implantations was the lowest in females at the dose level 60 mg/kg bw/day. The values of mating indexes showed that mating was not negatively affected by the test item treatment. Fertility indexes were lower at the dose level 60 mg/kg bw/day. The gestation index was comparable among the control and treated groups, but viability index on PND 4 was dose-dependently decreased. The lowest viability index was recorded at the dose level 120 mg/kg bw/day. Post-implantation and post-natal losses were significantly increased in females at the dose level 120 mg/kg bw/day.

The number of live born pups were significantly changed in treated females in comparison with the control females. A statistically significant decreased number of live born pups and number of pups at 4th day of lactation were recorded in females at the dose level 120 and 60 mg/kg bw/day. The stillborns were found only at the dose level 120 mg/kg bw/day. The mean weight of litters at birth, at PND 4 (postnatal day) and PND 13 was statistically significantly decreased at 120 and 60 mg/kg bw/day. A statistically significant reduction in the mean body weight of pups was recorded at the dose level 120 mg/kg bw/day; a statistically significant reduction in the mean weight of litters was recorded at the dose levels 60 and 120 mg/kg bw/day during all examination intervals. Macroscopical examination of pups showed an increased number with macroscopical findings at the dose level 120 mg/kg bw/day. Markedly smaller, thinner and less hairy pups were noted in two litters. An increased incidence of cannibalism and death of the pups was observed in females at the dose level 60 and 120 mg/kg bw/day. Twenty-six pups at the dose level 120 mg/kg bw/day and 4 pups at the dose levels 60 mg/kg bw/day could not be examined due to partial or total cannibalism. Thirteen pups at the dose level 120 mg/kg bw/day and 12 pups at the dose level 60 mg/kg bw/day were found dead during the lactation period. This effect – cannibalism, was not observed in the control group and the lowest group. Cannibalism was recorded sporadically at 60 mg/kg bw/day and markedly at 120 mg/kg bw/day. Therefore, dose dependency was recorded. Simultaneously it was found that mothers who cannibalized some pups were also good carers of their remaining pups. It could be speculated that only the altered pups were subject to cannibalism and that this phenomenon could therefore be used as indirect evidence that in litters affected by cannibalism, some type of developmental toxicity effects caused by the test substance treatment were present. On the other hand, the possibility that cannibalism could be a consequence of the test item toxicity to mothers cannot be ignored. Decreased food consumption and decreased body weight of mothers of the dose levels 60 and 120 mg/kg bw/day were recorded. Mothers who are stressed may show bizarre behaviour, including cannibalism. The toxic effect of the test item via lactation is unlikely because at the highest dose of 120 mg/kg bw/day there were also females in which cannibalism and/or death of pups were not recorded and the offspring were bred.

The NOAEL for reproduction was established as 120 mg/kg body weight/day. All changes in reproductive parameters observed in parental males and females at all dose levels were considered to be of no toxicological significance. The NOAEL for development was established as 30 mg/kg body weight/day. This judgement is based predominantly on increased cannibalism and on macroscopical findings (smaller, thinner pups, death of pups) of some pups at the dose levels 60 and 120 mg/kg bw/day.

Based on the physicochemical data and available in vivo toxicological data, there is systemic absorption after oral administration. For chemical safety assessment purposes, based on the physicochemical properties and information in the dossier, an oral absorption rate of 50% is accepted.

Absorption – dermal

In a dermal sensitization study (OECD 406/GLP) with Allyl Amyl Glycolate (CAS No. 67634-00-8) in ethanol, young adult Himalayan female guinea pigs were tested in a maximisation test. There were no positive skin reactions in any animals, so the substance is not sensitising.

Based on the physicochemical data, there is likely to be systemic absorption after dermal administration. The ECHA guidance criteria (Chapter R.7C) state that 10% dermal absorption is used when the molecular weight of the substance is >500 and the log Pow is <-1 or >4, otherwise 100% dermal absorption is used. In general, dermal absorption will not be higher than oral absorption, so for chemical safety assessment purposes a dermal absorption rate of 50% is accepted.

Absorption – inhalation

No inhalational toxicity study data is available. For chemical safety assessment purposes, an inhalation absorption rate of 100% is accepted using a conservative approach.

Distribution/Metabolism/Excretion

Based on the results of the combined repeated dose and reproduction/developmental toxicity screening test in male and female Wistar rats, there is wide distribution of the substance and metabolites throughout the body and potentially to the foetus. The liver is a target organ for toxicity in both sexes, with changes in biochemical parameters (liver enzymes), macroscopical and microscopical findings at 120 mg/kg bw/day. The MOA for allyl acetate-induced hepatotoxicity is known to be metabolism to allyl alcohol and then to acrolein, which leads to hepatocellular damage/death. Acrolein forms a conjugate with glutathione (GSH) in the liver (OECD, 2016). The conjugate is finally excreted in urine as 3-hydroxypropylmercapturic acid (3-HPM) through metabolic processing. This pathway is also likely for Allyl Amyl Glycolate.

OECD (2016). Case study on the use of an integrated approach to testing and assessment for hepatotoxicity of allyl esters. Series on Testing & Assessment No. 2