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Description of key information

There are two reliable 28-day oral toxicity studies in rats available for two different surrogates of the submission substance. Onyl effects observed were species specific effects on the liver as well as species and gender specific effects on the kidney. Mechanistic and histopahtological investigation were conducted to further support this evaluation (hepatic peroxisomal proliferation and alpha2u-globulin accumulation in kidneys). This findings were also obvious in 7-day dose range oral studies available for the submission substance and one other surrogate substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
There are two studies for surrogates of the submission substance according to OCED guideline 407 and conducted accoording to GLP (Klimisch 1). Furthermore there are two 7-day dose range findings studies (submission substance and surrogate) which have acceptable, well-documented study reports which meet basic scientific principles (Klimisch 2). The overall quality of the database is therefore high.

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

Additional information

A 90 day oral toxicity study in the rat has been proposed by the registrant of the surrogate substance 3,5,5-trimethylhexanoic acid according to the dissemination base of ECHA. As soon as these data are available the hazard assessment of the submission substance will be updated.

In a 28 day guideline study the test substance (purity 94.9%) was administered to male and female Wistar rats by gavage for 4 weeks at dose levels of 0 (vehicle control), 10, 50 and 200 mg/kg body weight/day asaqueous solution of 0.1% Cremophor EL. The test substance was a surrogate substance of the submission substance.Control and high dose groups consisted of each 10 animals per sex, whereas low and mid dose groups consisted of each 5 animals per sex. 5 animals per sex of all dose groups were sacrificed at the end of exposure (main groups). The remaining 5 animals per sex of control and high dose groups were maintained for another 14 dayswithout administration of the test substance (recovery groups).

Kidney effects were seen in males of all dose groups (dose-related effect). This was attributed to the sex and species-specific hydrocarbon-induced a2u globulin accumulation in the kidneys of male rats and regarded as not relevant for humans. No effects apart from nephrotoxicity in males and no effects at all were seen in females at 10 mg/kg bw/d.

At 50 mg/kg bw/d,fatty infiltration of liver cells (4 out of 10 animals) as well as alterations in clinical chemistry parameters and increased kidney weights were observed in females.

At 200 mg/kg bw/d, peripheral fatty infiltration of liver cells was increased in femals (all animals) and observed also in one male. In this dose group the following effects were observed additionally: decreased motor activity (females) and increased liver weights (both sexes) as well as alterations in clinical chemistry and urinalysis parameters (both sexes). Except the kidney lesions in male rats, no treatment-related effects were reported in the recovery group. Despite the effects observed in the 50 mg/kg dose group (fatty degeneration of the liver in female rats) this dose is considered to be the NOAEL:

The minimal liver effects in females at this dose are accompanied by signs of liver peroxisome proliferation, a species-specific effect in rodents. Moreover these effects were completely reversible. Increased kidney weights in females are also reversible and occured without any histological alterations in the kidneys, even at the highest dose tested. Therefore, the NOAEL of this study (BASF, 2002) is considered to be 50 mg/kg bw/d. This well performed guideline study (OECD 407) is considered to be of high reliability (RL1).

For the same surrogate substance slight maternal effects (decreased bw, increased liver weight) were observed in a one-generation rat study (reproduction/developmental toxicity screening test), but at higher doses (i.e. NOAEL = 79 – 228 mg/kg bw/day; Exxon, 1998; for further details please see the respective chapter).

 

A reliable 28-day oral toxicity study in rats has been performed with another surrogate substance of the submission substance (6-(Isononanoylamino)hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1)). The test animals received daily 0, 20, 100 or 500 mg test item per kg body weight for 28 consecutive days via oral gavage (OECD 407). The control group was treated with the vehicle only (distilled water). The Main Group animals were euthanized after 28 days of daily treatment. (Start of treatment, Day 0). Recovery animals, following the 28-day daily treatment were observed for a 14-day recovery period, than subjected to necropsy.

No clinical signs were observed and no lethality occurred during the 28-day treatment or recovery period.

There was no treatment-related effect on motor activity or in the functional observation battery tests across groups of treated male or female animals and no findings indicative of neurotoxicity were observed. Evaluation of the vaginal smears prior to necropsy showed the expected distribution of the oestrus cycle phases within the normal population of female Wistar rats.

There were no toxicologically significant changes in body weight, body weight gain or animal food consumption between the control and test item treated groups.

Minor variations, on occasion attaining statistical significance, were noted in the clinical pathology parameters (e.g. decreased blood glucose and urea concentration in males at 500 mg/kg). However, the changes were slight in magnitude, and/or were within the physiological range. Therefore these changes were not considered toxicologically significant.

No test item related observation was made at necropsy of animals following the 28-day treatment or 14-day recovery period.

At 500 mg/kg bw/day, the weights of kidneys (absolute and relative) were increased in males (approximately 25% on Day 28) and this was associated with hyaline deposition in tubular cells. No similar changes were observed in kidneys at 20 or 100 mg/kg bw/day (Low and Mid dose) or following recovery on Day 42.

At 500 mg/kg bw/day, the absolute and relative liver weights were higher than controls by approximately 30% in females and 10% in males following the 28-day treatment period, only and were associated with minimal periportal hepatocellular vacuolation of the liver (in 3 of 5 males and 4 of 5 females).

In a subsequent ex-vivo enyzme analysis of liver samples taken from control animals and high dose animals the increased liver weights could be ascribed to increased hepatic peroxisomal beta-oxidation (CN-palmitoyl-CoA-oxication values 1.9 and 1.8 fold higher in male and female rats respectively). Thus confirming that the effects on organ weights can be accounted for as species specific and therefore not adverse to humans.

In conclusion as effects observed in this 28 day oral gavage study (i.e. elevated liver weights in male and female animals and elevated kidney weights in male animals associated with hyaline deposition in tubular cells at the end of the treatment period; the effects on the liver weights were consistent with microscopic findings in high dose group, but reversible within the 14 day recovery period) were due to species specific induction of hepatic peroxisomal beta-oxidation (liver effects) or alpha2u globulin accumulation (kidney effects in male rats) these effects wer not attributed as adverse (as not relevant for human risk assessment) and thus a NOAEL at the highest dose group was set (i..e. 500 mg/kg bw/day).

Furthermore there are two 7 day oral dose range finding studies available. One study was performed with the submission substance (6-(Isononanoylamino)hexanoic acid) and another study was performed with a surrogate of the submission substance (6-(Isononanoylamino)hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:3)).

The objective of these studies was to determine the Maximum Tolerated Dose (MTD) and to obtain preliminary information on the toxicity of the test item when administered to Wistar rats at three dose levels for up to 7 days by oral gavage.

In each study during the Phase 1 the dose of 1000 mg/kg was tested as a single gavage to two male and two female rats. The animals survived the treatment, no clinical signs were recorded on the treatment day or subsequent 6-day observation period, no adverse effect on body weight or food consumption was found and no remarkable observation was noticed during the scheduled necropsy. Based on the results the Phase 2 started at 3 dose levels of 62.5, 250 and 1000 mg/kg bw/day.

At necropsy of the Phase 1 animals, there was no evidence of test item-related macroscopic findings.

For Phase 2 the test item was either formulated in distilled water (surrogate) or PEG 400 and 6% ethanol (submission substance) and the control groups was treated each with the respective vehicle only.

There were no clinical signs or unscheduled mortality during the study. All animals were symptom-free after a 7-day daily treatment at 62.5, 250 and 1000 mg/kg bw/day, during Phase 2 of the study.

There were no toxicologically significant or adverse effects observed on the animal body weights or food consumption.

Hematology and clinical chemistry parameters were mostly within normal range. Observed changes were most probably considered correlated to the species specific changes (i.e. increased hepatic beta- oxidation anda2u globulin accumulation in kidneys).

At necropsy of the Phase 2 animals the effects on liver and kidneys became evident (e.g. pale discolouration of all lobes in the liver and increased liver weights male and female animals; bilateral pale discolouration of the kidneys was seen in males accompanied by a slight increase in organ weight; with most effects starting at mid dose group but being most evident in high dose group).

In subsequent ex-vivo enyzme analyses of liver samples taken from control animals and high dose animals the increase of hepatic peroxisomal beta-oxidation was confirmed for both test materials. Therefore the increased liver weights could be ascribed to this species specific effect and thus not adverse to humans.

In conclusion, the NOAEL for both test items under the conditions of this study was considered to be 1000 mg/kg bw/day (as only species specific effects were observed up to this dose).

Evidence for Peroxisomal Proliferation as mode of action for liver toxicity

In the following studies enzyme analysis were performed:

- 7 -day dose range finding study for the submission substance

- 7 -day dose range finding study for a surrogate of the submission substance

- 28 -day oral toxicity study (OECD 407) for the same surrogate of the submission substance

- 28 -day oral toxicity study (OECD 407) for another surrogate of the submission substance

The analysis determined the activity of the cyanide insenstitive palitoyl CoA oxidation (PCO), which is a marker for peroxisomal beta-oxidation. In all studies the PCO was increased at least 1.2 up to 3.2 fold, thus correlating the observed liver effects with hepatic peroxisomal proliferation.

Evidence for alpha 2u-globulin accumulation as mode of action for kidney toxicity

Histopathological investigations in the 28 -day oral toxicity studies revealed the alpha 2u-globulin accumulation in kidneys of male rats and thus verifying the assumptions that kidney effects are du to the alpha 2u-globulin-induced nephropathy which is specific for male rats and thus not relevant for humans. Any effects on female kidneys (e.g. slight elevation of organ wieght) were always reversible and without concomitant histological alterations.

Remark: Even though the composition of test material was slightly different in the 28-day repeated dose study and the one 7-day repeated dose study (i.e.6-(Isononanoylamino)hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) versus 6-(Isononanoylamino)hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:3)) the comparable effects observed reveal that the varying amounts of 2,2’,2’’-nitrilotriethanol (TEA) are not affecting the toxicological characteristics of the chemical compound.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
A reliable study with lowest NOAEL for surrogate of submission substance

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
no study required as the oral application route is expected to be the relevant exposure route for humans, due to the physical-chemical properties of the submission substance

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
no study required as the oral application route is expected to be the relevant exposure route for humans, due to the physical-chemical properties of the submission substance

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
no study required as the oral application route is expected to be the relevant exposure route for humans, due to the physical-chemical properties of the submission substance

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
no study required as the oral application route is expected to be the relevant exposure route for humans, due to the physical-chemical properties of the submission substance

Justification for classification or non-classification

No relevant toxic effects were observed in oral repeated dose toxicity studies in rats (highest dose tested 200, 500 or 1000 mg/kg bw/day in OECD 407 and 7 -day dose range findings studies performed with the submission substance or surrogates). No data on repeated dose toxicity after inhalation or dermal exposure are available. Therefore the submission substance does not have to be classified for specific target organ toxicity – repeated exposure or repeated dose toxicity according to Regulation (EC) No 1272/2008 or to Council Directive 67/548/EEC.