Hexanoic acid, 2-ethyl-, C12-15-alkyl esters

Endpoint summary

Description of key information

Study performed to recognised testing guideline and GLP.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 August 2001 - 3 September 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

October 2008

Deviations:

yes

Remarks:

no detailed clinical observations made; no neurobehavioural tests performed; prostate, testes and epididymides not weighed.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): isononyl isononanoate
- Physical state: colourless liquid
- Analytical purity: no data
- Storage condition of test material: at room temperature

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France, Saint Aubin-lès-Elbeuf, France
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 204 g (males), 169 g (females)
- Fasting period before study: no
- Housing: the animals were housed 2 per cage, according to sex and dose group, in suspended wire-mesh cages (43.0 cm x 21.5 cm x 18.0 cm). A metallic tray containing autoclaved sawdust (SICSA, Alfortville, France) was placed under each cage and the sawdust changed once per week.
- Diet: A04 C pelleted maintenance diet, batch No. 90528 (U.A.R., Villemoisson-sur-Orge, France), ad libitum
- Water: tap water filtered using a 0.22 micron filter, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 04 Aug 2001 To: 2-3 Sep 2001

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: the dosing solutions were made daily by mixing the test substance with the vehicle to the required concentration of 20, 60 and 200 mg/mL and homogenising the solutions using a magnetic stirrer. The solutions were stirred continously during the dosing procedure. Doses were adjusted acording to the most recently recorded body weight of each animal.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 107H1649 (Sigma, Saint-Quentin-Fallavier, France)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

29-30 days

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and signs of morbidity, at least once daily for clinical signs

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 (prior to administration), 8, 15, 22 and 29 (prior to sacrifice)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, based on intake recorded once per week
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 of the study period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: yes, overnight (at least 14 hours)
- How many animals: all the animals in the control group and treatment groups
- Parameters examined: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes (platelets), total leucocytes, differential leucocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes), reticulocytes, prothrombin time, activated partial thromboplastin time, fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28 of the study period
- Animals fasted: yes, overnight (at least 14 hours)
- How many animals: all the animals in the control group and treatment groups
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total protein, albumin, albumin/globulin ratio, cholesterol, triglycerides, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase

URINALYSIS: Yes
- Time schedule for collection of urine: Day 28 of the study period
- Metabolism cages used for collection of urine: yes
- Animals fasted: yes, overnight (at least 14 hours)
- Parameters examined: volume, pH, specific gravity, proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, sediment (microscopic cytology determining leucoytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals, cells), appearance, colour

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and signs of morbidity, at least once daily for clinical signs

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 (prior to administration), 8, 15, 22 and 29 (prior to sacrifice)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, based on intake recorded once per week
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 of the study period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: yes, overnight (at least 14 hours)
- How many animals: all the animals in the control group and treatment groups
- Parameters examined: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes (platelets), total leucocytes, differential leucocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes), reticulocytes, prothrombin time, activated partial thromboplastin time, fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28 of the study period
- Animals fasted: yes, overnight (at least 14 hours)
- How many animals: all the animals in the control group and treatment groups
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total protein, albumin, albumin/globulin ratio, cholesterol, triglycerides, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase

URINALYSIS: Yes
- Time schedule for collection of urine: Day 28 of the study period
- Metabolism cages used for collection of urine: yes
- Animals fasted: yes, overnight (at least 14 hours)
- Parameters examined: volume, pH, specific gravity, proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, sediment (microscopic cytology determining leucoytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals, cells), appearance, colour

NEUROBEHAVIOURAL EXAMINATION: No

Statistics:

Body weight, food consumption, hematology, blood biochemistry, urinalysis and organ weight data were analysed for statistical significance. The tests were applied according to the flow chart attached in 'Attached background material'.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: 4/10 females died/killed prematurely

Mortality:

mortality observed, treatment-related

Description (incidence):

1000 mg/kg bw/day: 4/10 females died/killed prematurely

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: reduced body weight gain

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: reduced APTT level (males, non-adverse); increased monocyte ceel count (non -adverse)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw/day: increased ALP and ALAT (females) (non adverse)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: increased volume

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/day: increased kidney weight (males, non-adverse)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/day: accentuated hepatic lobular pattern, liver paleness, enlarged liver (females) (non-adverse)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/day: steatosis (non-adverse)

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
4/10 females in the high-dose group died or were killed prematurely (Day 9, 10, 13 and 25, respectively). Three of the four animals showed signs of poor clinical condition prior to death, like emaciation, locomotor difficulties, dyspnea, coldness to the touch and piloerection. The post-mortem examination showed liver paleness, with steatosis and diffuse hepatocellular hypertrophy; contracted spleen; vacuolated cortical tubular epithelium in the kidneys; and porphyrin deposition in the Harderian gland in at least 3/4 females. Although no direct cause of mortality or a moribund condition could be found, the general poor condition and post-mortem findings are considered to be toxicologically relevant. 1/10 females in the mid-dose group died on Day 11. The necropsy showed that the rat had a reduced spleen size; colour changes, steatosis and diffuse hepatocellular hypertrophy in the liver; and cortical cell hypertrophy in the adrenals. No clinical signs were observed prior to death. Similar effects in the liver were also observed in the moribund female rats of the high-dose group, however, these are considered to be adaptive effects. As all of the findings were considered to be adaptive, the death of the single mid-dose female is considered to be treatment-related, but not toxicologically relevant.

In 1, 3, and 7 females in the low-, mid- and high-dose group, respectively, some hair loss and/or alopecia was noted. This might be related to the accidental dermal exposure of smalll amounts during handling and dosing of the animals. The substance is irritating to the skin in high doses (as noted under 7.5.3, 14-day dermal repeated dose toxicity study) and a small amount on the gloves of handlers may cause irritation. This effect is not seen as toxicologically relevant for hazard evaluation of the oral route.

BODY WEIGHT AND WEIGHT GAIN
In males administered the highest dose, the body weight was significantly lower on Day 8 and Day 29 (-13% at the end of the study period), compared with the control group. In females of the high-dose group, the lower body weight was significant only on Day 8, but also marked on Day 29 (-11%, compared with the control group) (see Table 1 under 'Any other information on results incl. tables'). The wide standard deviation indicates that some of the animals were emaciated, most likely the females that died or were sacrificed prematurely. However, no individual data was reported. The body weight gain was reduced in males (significantly) and females (non-significantly) during the whole study period, compared with the body weight gain of control group (see Table 2 under 'Any other information on results incl. tables'). The effects on body weight (gain) are considered to be toxicologically relevant. Slightly lower body weight gain was noted in the low- and mid-dose groups, but as these were 3-5% and not dose-related, they are not considered to be toxicologically relevant.

FOOD CONSUMPTION
During week 1, the food intake was significantly reduced in the high-dose groups (-17% for males and -31% for females, respectively, compared with the control group). However, the food intake during the rest of the study period was comparable in all groups, and there was no significant difference in food consumption between the control group and treatment groups as calculated over the whole study period.

HAEMATOLOGY
An increase in monocyte level in the males of the mid- and high-dose groups of 64 and 67%, respectively, was observed, but not significantly and with large standard deviations. In the females, the increase was 47% in the mid-dose group (no statistical significance) and 110% in the high-dose group with statistical significance. The mean values were within the historical data range of 0.11 - 0.83 G/L for males and 0.07 - 0.58 G/L for females, although not all individual data were reported (see Table 3 under 'Any other information on results incl. tables'). As there were no effects on other individual lymphocyte counts, nor on the total lymphocyte count, this effect is not considered to be toxicologically relevant. The level of activated partial thromboplastin time (APTT) was significantly reduced in males of the high-dose group. This effect was not seen in females, and most of the individual results were within the historical data range. Therefore the effect is not considered to be toxicologically relevant. Significant effects observed in the low- or mid- dose group only are viewed as incidental occurrences due to a lack of dose-response relation.

CLINICAL CHEMISTRY
The alanine aminotransferase (ALAT) levels were increased non-significantly in the mid-dose group and significantly in the high-dose group in males and females (see Table 4 under 'Any other information on results incl. tables'). The aspartate aminotransferase (ASAT) levels were increased non-significantly in the mid-dose males, and significantly in the mid-dose females and the high-dose group in males and females. A significant increase in the alkaline phosphatase (ALP) level was noted in high-dose females only. The increased activity of these hepatic enzymes indicates an increase in hepatic metabolism due to the treatment and might be related to the pathological changes observed in the liver. The glucose level was significantly decreased at all dose levels in males and females. According to the authors, almost all the values fell within the historical control range of the laboratory (5.45 - 9.84 mmol/L for males and 4.81 - 8.90 mmol/L for females), however the individual results were not included in the study report. As the effect is dose-related, it is considered to be related to the liver changes. Significant increases in the urea levels were noted in males and females in the mid-and high-dose groups. According to the authors, almost all thevalues fell within the historical control range of the laboratory (3.4 - 7.3 mmol/L for males and 3.9 - 8.0 mmol/L for females), however the individual results were not included in the study report. The increase in urea levels is considered to be toxicologically relevant at the highest dose level, as histopathological effects were noted in the kidneys. A significant increase in the inorganic phosphatase level was noted in the low- mid- and high dose females, as well as in the high-dose males. The control value in the females was low compared with the historical control values from the laboratory, which may have contributed to the difference. According to the authors, all the values fell within the historical control range of the laboratory (2.40 - 3.25 mmol/L for males and 1.66 - 2.77 mmol/L for females), however, the individual results were not included in the study report. The toxicological significance of this effect is unclear. The calcium levels were increased significantly in all dose groups in males and females. However, this is not considered be of toxicological significance as the increases were less than 10%. Significant effects observed in the low- or mid- dose group only were viewed as incidental occurrences.

URINALYSIS
The urinary volume was increased significantly in low-dose females, and in high-dose males and females (see Table 5 under 'Any other information on results incl. tables'). While the increase in the low-dose females is most likely incidental due to the lack of similar effects in the mid-dose animals, the results observed in the high-dose animals are considered to be toxicologically relevant.

ORGAN WEIGHTS
A dose-related increase in absolute and relative liver weight was observed at all dose levels compared with the control groups, with significant difference from the control group in the mid- and high dose groups for males and females (see Table 6 under 'Any other information on results incl. tables'). The increase in lvier weight is considered to be treatment-related and correlated with macroscopic and microscopic changes noted in the liver. The relative kidney weight was significantly increased compared with the controls at all dose levels for males and females, while the absolute weight was increased significantly for low-dose females only. The effects noted in males at all dose levels are considered to be related to the histopathological findings, while for females a possible histopathological relation was noted at the mid- and high dose levels. Reduced spleen weight and thymus weight noted in high-dose females were related to the general poor health condition of some individuals in this group. The decrease in absolute ovary weight in high-dose females and increase in relative testes weight in high-dose males compared with the control groups, respectively, is considered to be incidental as the changes were less than 10%.

GROSS PATHOLOGY
All the results include animals examined at scheduled and unscheduled necropsy. A grey/green colouration of the kidney was noted in 0/10, 1/10, 3/10 and 5/10 males in the control, low-, mid- and high-dose group, respectively, and in 0/10, 1/10, 2/10 and 1/10 females in the control, low-, mid- and high-dose group, respectively (see Table 7 under 'Any other information on results incl. tables'). A dose-related increase in the number of animals with enlarged liver was observed. An accentuated lobular pattern was reported in 0/10, 4/10, 4/10 and 8/10 males, and 0/10, 7/10, 9/10 and 3/10 females in the control, low-, mid- and high-dose group, respectively. The incidence of liver paleness was notable in females, with 0/10, 4/10, 3/10 and 7/10 in the control, low-, mid- and high-dose group, respectively, while it was observed in 2/10 males in the high-dose group. The effects seen in the kidneys and liver males and females are correlated with microscopic effects described below. The reduced spleen- and thymus size noted in high-dose females is considered to be secondary to the poor general health condition of these animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
All the results include animals examined at scheduled and unscheduled necropsy. Steatosis was observed in 5/10 females in the control group and in a majority of male and female rats at all dose levels (see Table 8 under 'Any other information on results incl. tables'). The number of cases and the severity increased with increasing dose, indicating a treatment-related effect. Diets high in fatty acids are known to induce steatosis in rats and the effect is therefore considered to be an adaptive rather than an adverse effect (Buettner, R. et al. Defining high-fat-diet rat models: metabolic and molecular effects of different fat types. J Molec Endocrinol (2006), 6: 485-501). Hepatocellular hypertrophy was noted in 0/10, 0/10, 9/10 and 9/10 males in the control, low-, mid- and high-dose group, respectively, and in 0/10, 0/10, 10/10 and 10/10 females in the control, low-, mid- and high-dose group, respectively. This effect is an adaptive effect of the high hepatic load caused by exposure to the test substance (Bär, A. Charateristics and significance of D-tagatose-induced liver enlargement in rats: an interpretative review. Reg Toxicol Pharmacol 1999, 29:S83 - S93). In males, acidophilic globules in the cortical tubular epithelium of the kidney was observed in 10/10 rats in all the dose groups. The incidence and severity is considered to be caused by the increased production of alpha-2-microglobulin. This effect is only observed in male rodents and is not relevant to humans. The incidence of renal vacuolated cortical tubular epithelium in 2/10 female rats in the mid-dose group and 6/10 in the high-dose group is a treatment-related effect that is known to occur in rats fed high-fat diets (Altunkaynak, M.E. et al. The effects of high-fat diet on the renal structure and morphometric parametric of kidneys in rats. J Anat 2008, 212:845 - 852). Cases of contracted spleen and lymphoid depletion of the thymus noted in high-dose females is considered to be secondary to the poor general health condition of these animals.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Table 1: Body weight

	Group (mg/kg bw/day)
	Males				Females
Day	Control	100	300	1000	Control	100	300	1000
1	204 ± 11.0	203 ± 11.1	206 ± 10.6	204 ± 9.3	168 ± 9.3	169 ± 8.3	167 ± 6.3	171 ± 8.9
8	263 ± 15.2	259 ± 14.8	261 ± 11.6	243 ± 26.2*	194 ± 10.7	195 ± 10.9	190 ± 8.9	173 ± 16.7**
15	308 ± 19.9	307 ± 20.5	305 ± 14.7	289 ± 30.2	216 ± 13.9	221 ± 17.2	212 ± 9.9	198 ± 28.3
22	337 ± 23.5	332 ± 31.0	329 ±16.5	317 ± 27.3	233 ± 17.5	241 ± 21.2	231 ± 8.6	223 ± 25.8
29	355 ± 26.1	346 ± 36.2	337 ± 19.6	310 ± 29.3**	240 ± 16.8	250 ± 23.5	235 ± 9.3	214 ± 36.6

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 2: Body weight Day 1 - 28

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Total body weight gain (g)	151	143	131	106	72	81	68	43
Body weight change Day 1 - 28 (relative)	x 1.7	x 1.7	x 1.6	x 1.5	x 1.4	x 1.5	x 1.4	x 1.3
Difference from control (%)	-	-3	-5	-13	-	-4	-2	-11

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 3: Selected haematology results

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Monocytes (G/L)	0.28 ± 0.094	0.32 ± 0.155	0.46 ± 0.332	0.47 ± 0.258	0.17 ± 0.077	0.19 ± 0.077	0.25 ± 0.104	0.36 ± 0.095**
WBC	10.78 ± 1.97	8.45 ± 2.04	9.84 ± 3.75	10.36 ± 2.07	6.28 ± 1.26	6.74 ± 1.63	7.57 ± 2.30	6.00 ± 1.19
APTT	23.2 ± 3.70	24.7 ± 3.67	24.5 ± 5.80	17.9 ± 4.51*	16.1 ± 2.71	15.4 ± 1.32	14.1 ± 1.43	15.5 ± 3.15

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 4: Selected clinical chemistry results

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Calcium (mmol/L)	2.66 ± 0.05	2.78 ± 0.07**	2.75 ± 0.07*	2.74 ± 0.08*	2.77 ± 0.06	2.62 ± 0.06*	2.67 ± 0.09**	2.62 ± 0.08**
Inorganic phosphate (mmol/L)	2.69 ± 0.197	2.69 ± 0.157	2.73 ± 0.100	2.94 ± 0.192**	1.98 ± 0.255	2.30 ± 0.279*	2.41 ± 0.123**	2.58 ± 0.223**
Glucose (mmol/L)	7.17 ± 0.592	5.85 ± 0.436**	5.50 ± 0.190**	5.49 ± 0.684**	7.08 ± 0.806	6.13 ± 0.489*	5.70 ± 0.885**	5.81 ± 1.193*
Urea (mmol/L)	3.5 ± 0.59	4.1 ± 0.59	4.5 ± 0.63**	4.3 ± 0.48*	5.1 ± 0.48	6.0 ± 1.10	6.5 ± 1.60*	6.6 ± 1.02*
ALP (IU/L)	313 ± 35.6	312 ± 62.6	279 ± 51.4	338 ± 111.8	190 ± 40.4	244 ± 73.5	316 ± 118.7**	364 ± 158.9**
ASAT (IU/L)	57 ± 6.3	56 ± 7.2	69 ± 13.9	86 ± 29.1**	59 ± 15.2	50 ± 18.9	75 ± 17.9	120 ± 41.1**
ALAT (IU/L)	14 ± 2.2	18 ± 4.5	21 ± 8.0	42 ± 25.9**	14 ± 5.3	24 ± 10.1	35 ± 12.3**	48 ± 15.8**

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

Table 5: Urinary volume results

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Volume (mL)	14 ± 11.2	14 ± 7.7	19 ± 7.8	28 ± 12.0*	11 ± 4.2	23 ± 9.0*	17 ± 14.1	27 ± 13.6*

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

Table 6: Selected organ weights

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Kidney, absolute (g)	2.62 ± 0.249	3.09 ± 0.320*	3.00 ± 0.287	2.98 ± 0.536	1.79 ± 0.168	2.01 ± 0.189*	1.85 ± 0.95	1.91 ± 0.255
Kidney, relative (%)	0.786 ± 0.047	0.965 ± 0.097**	0.964 ± 0.084**	1.03 ± 0.098**	0.801 ± 0.061	0.882 ± 0.062**	0.877 ± 0.043*	0.990 ± 0.052**
Liver, absolute (g)	10.9 ± 1.84	11.26 ± 1.47	13.02 ± 0.820*	16.20 ± 3.60**	7.32 ± 0.750	9.69 ± 1.42*	11.35 ± 1.46**	13.60 ± 4.48**
Liver, relative (%)	3.27 ± 0.385	3.49 ± 0.142	4.19 ± 0.330**	5.62 ± 1.01**	3.27 ± 0.247	4.23 ± 0.309	5.38 ± 0.559**	7.02 ± 2.22**
Spleen, absolute (g)	0.651 ± 0.127	0.626 ± 0.139	0.580 ± 0.117	0.502 ± 0.102*	0.534 ± 0.097	0.538 ± 0.102	0.427 ± 0.060*	0.333 ± 0.115*
Spleen, relative (%)	0.195 ± 0.032	0.194 ± 0.030	0.185 ± 0.029	0.174 ± 0.023	0.239 ± 0.042	0.235 ± 0.033	0.203 ± 0.028	0.170 ± 0.042**
Thymus, absolute (g)	0.469 ± 0.152	0.452 ± 0.104	0.426 ± 0.075	0.360 ± 0.071	0.412 ± 0.058	0.411 ± 0.058	0.328 ± 0.058*	0.260 ± 0.132**
Thymus, relative (%)	0.140 ± 0.042	0.140 ± 0.024	0.137 ± 0.022	0.127 ± 0.030	0.184 ± 0.021	0.180 ± 0.013	0.155 ± 0.021*	0.130 ± 0.050
Ovaries, absolute (g)	-	-	-	-	0.121 ± 0.017	0.129 ± 0.017	0.117 ± 0.012	0.099 ± 0.016*
Ovaries, relative (%)	-	-	-	-	0.054 ± 0.007	0.056 ± 0.006	0.056 ± 0.006	0.051 ± 0.007
Testes, absolute (g)	3.25 ± 0.258	3.15 ± 0.335	3.18 ± 0.193	3.11 ± 0.328	-	-	-	-
Testes, relative (%)	0.978 ± 0.089	0.985 ± 0.107	1.02 ± 0.071	1.09 ± 0.094*	-	-	-	-

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 7: Gross pathology results (incl. deaths/interim sacrifices)

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Liver, accentuated lobular pattern	0/10	04-Oct	04-Oct	08-Oct	0/10	07-Oct	09-Oct	03-Oct
Liver, enlarged	0/10	01-Oct	01-Oct	06-Oct	0/10	0/10	01-Oct	05-Oct
Liver, paleness	0/10	0/10	0/10	02-Oct	0/10	04-Oct	03-Oct	07-Oct
Kidney, grey/green colour	0/10	01-Oct	03-Oct	05-Oct	0/10	01-Oct	02-Oct	01-Oct
Spleen, reduced in size	0/10	0/10	0/10	01-Oct	0/10	0/10	01-Oct	04-Oct
Thymus, reduced in size	0/10	0/10	0/10	0/10	0/10	0/10	0/10	02-Oct

Table 8: Histopathology results

	Group (mg/kg bw/day)
	Males				Females
	Control	100	300	1000	Control	100	300	1000
Liver, moderate to severe steatosis	0/10	05-Oct	07-Oct	08-Oct	05-Oct	10-Oct	10-Oct	10-Oct
Liver, slight to severe hepatocellular hypertrophy	0/10	0/10	09-Oct	09-Oct	0/10	0/10	10-Oct	10-Oct
Kidney, acidophilic globules in cortical tubular epithelium, minimal to slight	0/10	10-Oct	10-Oct	10-Oct	0/10	0/10	0/10	0/10
Kidney, vacuolated cortical tubular epithelium, minimal to moderate	0/10	0/10	0/10	0/10	0/10	0/10	02-Oct	06-Oct
Spleen, contracted	0/10	0/10	0/10	0/10	0/10	0/10	0/10	05-Oct
Thymus, lymphoid depletion	0/10	0/10	0/10	0/10	0/10	0/10	0/10	07-Oct

Conclusions:

The test material 28 day repeat dose NOAEL (males and females) was determined to be 300 mg/kg bw/day under the conditions of the test.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Read across from similar substance.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Full information on the read-across approach is contained within the attached justification document.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source and target substances are composed of the same chemical groups and are bonded together in functionally the same way. The source substance and target substances have low water solubility, high partition coefficient, and are in the physical form of a liquid with a neutral pH. Available toxicity information on the source and target substances indicates that they behave in substantially similar ways in the body. The potential for repeat dose toxicity is therefore the same in all substances.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source substance and target substances all have branched acid and alcohol groups, the alcohol parts of all three substances are also branching – each substance has an alcohol branch in which the chains are present as a variety of branched species. The main difference is that in the case of 3,5,5-trimethylhexyl 3,5,5-trimethylhexanoate, both the starting acid and alcohol used to synthesise it are both mono-constituent substances producing a mono-constituent ester, whereas all the target substances are UVCBs because one of the starting components being a UVCB. The UVCB components of C12-15 Alkyl Ethylhexanoate and Isodecyl 3,5,5-trimethylhexanoate/Isotridecyl 3,5,5-trimethylhexanoate are 2-ethylhexanoic acid and 3,5,5-trimethyl hexanoic acid respectively. In addition, the branching in the target substances is due to the alcohol chain as in each target substance this chain is present as saturated isomers with various branching chains. Another significant difference is that C12-15 Alkyl Ethylhexanoate has a greater variation, as the alcohol contains twelve to fifteen carbons in total, whereas in Isodecyl 3,5,5-trimethylhexanoate/Isotridecyl 3,5,5-trimethylhexanoate there are ten and thirteen carbons in the alcohol groups respectively.
The number of carbons in the alcohol part of the esters in all cases has an odd number, which can affect toxicity. There is one exception, Isodecyl 3,5,5-trimethylhexanoate, which has an even amount. Due to the variable amount of carbons present in C12-15 Alkyl Ethylhexanoate, this substance can also contain an even amount of carbons.
The acid part of the source substance contains an odd number of carbons, and this is an attribute shared by two of the target substances Isodecyl 3,5,5-trimethylhexanoate and Isotridecyl 3,5,5-trimethylhexanoate. Only the target substance C12-15 Alkyl Ethylhexanoate has an even number of carbons.
Neither the source substance nor the target substances have hydrogen donor or hydrogen acceptor sites. The source substance and the target substances are fully saturated, with two hydrogens for every carbon. The molecular weights are very similar (all in the range of 300-355 Da), and this means that the physical-chemical properties of the substances are very much alike (see Table 2), in particular:
• All are water insoluble (<0.2 mg/mL)
• All are liquids at room temperature with melting points <-20°C
• All have a boiling point or exothermic reaction at >300°C
• All have a relative density of 0.85-0.86 g/cm3
• All have high partition coefficients (Log KoW) of >7.2 at 35 °C pH 7
The source substance and the target substances are also unclassified under 1278/2008 CLP regulation for both skin and eye irritation/corrosion. In addition, all the substances have an acute oral toxicity of >4300 mg/kg bw, which also means they are unclassified. This, as well as the similarities in acute toxicity, indicate that they will have similar bioavailability.


3. ANALOGUE APPROACH JUSTIFICATION
Due to the similarities of the source and target substances with regards to branching, odd and even numbered alkyl and acid chains, the target substances are expected to behave in a substantially similar manner in vivo.
The target substances are therefore predicted to induce similar levels of systemic toxicity effects in the OECD 407 study when conducted in the rat. While systemic toxicity was observed in the source study, the NOAEL of 300 mg/kg bw/day is not sufficient to fulfil the requirements for classification according to the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

4. DATA MATRIX
Full information on the read-across approach is contained within the attached justification document.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1

Organ:

kidney

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

Justification for classification or non-classification

The substance failed to induce systemic toxicity sufficient to meet the cirteria of the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).