Naphtha (Fischer-Tropsch), light, C4-10-branched and linear

Endpoint summary

Description of key information

- Oral: NOAEL is considered to be 200 mg/kg bw based on results of a reliable OECD 408 study for 'Naphtha (Fischer-Tropsch), light, C4-C10 branched and linear'. ´

- Inhalation (based on results of a reliable OECD 413 study for a related substance, covering the carbon numbers in the range C7-C10): Systemic LOAEL = 6646 ppm (24,300 mg/m3) based on increased liver weight and red facial staining; NOAEL = 2220 ppm (8200 mg/m3); neurotoxicity NOAEL = 6646 ppm (24,300 mg/m3).

- Dermal: Contact with skin is not considered to be the primary route of exposure. 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear' contains volatile components and its vapour pressure indicates that inhalation may be the significant pathway for human exposure.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2007 - May 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: six to eight weeks
- Weight at study initiation:it averages for males 168,2 g and for females 149,8 (these means are calculated from data of first study day for all groups tested)
- Fasting period before study: no
- Housing: in groups of three or four by sex in polypropylene cages with stainless steel mesh lids and solid bases containing softwood chip bedding in a single air-conditioned room
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

The test material was administered daily by gavage using stainless steel cannula attached to a disposable plastic syringe. Control animals were treated only with vehicle in an identical manner.

PREPARATION OF DOSING SOLUTIONS: test material was prepared at the appropriated concentrations as solutions in Arachis oil BP, which was served as vehicle and as common control.
- Rate of preparation of dosing solution (frequency): weekly
- Mixing appropriate amounts with (Type of food): vehicle
- Storage temperature of food: at +4°C in the dark


VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis oil BP was chosen as a suitable diluter for hydrocarbons. Arachis oil is a monounsaturated fat, wide used as a most neutral food component in a variety of diets, also known for its high smoke point and stability. The oil ensures an optimal intake of test material in gastrointestinal tract.
- Concentration in vehicle: 0; 12,5; 50 and 188 mg/ml
- Amount of vehicle (if gavage): 4ml/kg/day
- Lot/batch no. (if required): data not avilable
- Purity: data not available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test material formulation were taken and analysed for concentration of 'Naphtha (Fischer-Tropsch), light, C4—10-branched and
linear'. The concentration of test material in formulations was determined by gas chromatography (GC) using an external standard technique. The analytical method has been satisfactorily validated in terms of linearity, specificity and
accuracy for the purposes of the study.The results indicate that the prepared formulations were within ± 10% of the nominal concentration and were stable for at least fourteen days.

Duration of treatment / exposure:

1. Non-recovery groups of animals: ninety consecutive days/oral gavage.
2. Two recovery groups were treated with the high dose (1000 mg/kg/day) or the vehicle alone for ninety consecutive days and then
maintained without treatment for a further twenty-eight days/oral gavage.

Frequency of treatment:

daily at the same timepoint

Remarks:

Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
200 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
750 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

- ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats at dose levels of 50, 200 and 750 mg/kg/day
- control group: ten males and ten females with vehicle alone (Arachis oil BP)
- two recovery groups, each of ten males and ten females, were treated with the high dose (750 mg/kg/day) or the vehicle alone

Dose Level Animal Numbers
------------------------------------------------
(mg/kg/day) Male Female
------------------------------------------------
Control 0 10 10
Recovery Control 0 10 10
Low 50 10 10
Intermediate 200 10 10
High 750 10 10
Recovery High 750 10 10

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were randomly assigned to the control and treatment groups according to OECD Guidline 408 "Repeated Dose 90-Day Oral Toxicity in
Rodents"

- Dose selection rationale: were ascertained from the preliminary fourteen day repeated dose oral (gavage) range-finder study in the rat: test material in the concentrations 300 mg/kg/day and 1000 mg/kg/day as well as control group treated with vehicle alone was administered daily. Increased salivation which was detected for males and females at the 1000 mg/kg/day and for males at the 300 mg/kg/day as well as individual incident of palish kidney and sloughing on the non-glandular region of stomach from the treatment group of 1000 mg/kg/day were the reason to determine the dose levels for the ninety day study as high dose 750 mg/kg/day, intermediate dose 200 mg/kg/day and low dose 50 mg/kg/day.

- Rationale for animal assignment (if not random): randomized

- Rationale for selecting satellite groups: randomized

A common control group, treated only with vehicle alone, is needed to exclude any effect of vehicle and make possible to interpret the effects of test material. The aim of recovery groups is to observe reversibility or persistence of any toxic effects.

- Post-exposure recovery period in satellite groups: 28 days

- Section schedule rationale (if not random): randomized

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change
immediately before dosing, post dosing and one and five hours after dosing during the working week; immediately before dosing, post dosing and one hour after dosing at weekends and public holidays. During the treatment-free period, animals were observed twice daily (once daily at weekends)

BODY WEIGHT: Yes
- Time schedule for examinations: on day 1 of study and at weekly intervals thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No feeding study but oral gavage.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD CONSUMPTION: was recorded for each cage group at weekly intervals throughout the study (calculated in g/animal/day).

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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No drinking water study but oral gavage.
- Time schedule for examinations:

WATER CONSUMPTION: daily by visual inspection.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: the eyes were examined pretreatment and before termination of treatment (during Week 12).
- Dose groups that were examined: non-recovery control and non-recovery high dose animals.

Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following
pupil dilation with 1.0% “Tropicamide” solution, detailed examination of the internal structure of the eye using a direct
ophthalmoscope was performed.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period (day 90) and on all recovery group animals at
the end of the treatment-free period (day 118).
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all animals from each test and control group as well as all recovery group animals.
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period (day 90) and on all recovery group animals at the end of the treatment-free period
(day 118).
- Animals fasted: No
- How many animals: all animals from each test and control group as well as all recovery group animals
- Parameters checked in table [No.2] were examined.The parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-
coagulant.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
All animals were observed for signs of functional/behavioural toxicity. During week 12 functional performances tests were also
performed on all non-recovery animals together with an assessment of sensory reactivity to different stimuli.

- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter.
- Dose groups that were examined: only non-recovery (without 28 days treatment-free period) animals.
- Battery of functions tested: sensory activity (Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex
Finger approach) grip strength and motor activity.
- Parameters checked in table [No.3] were examined using a purpose built arena.

Sacrifice and pathology:

GROSS PATHOLOGY: (Yes) On completion of the dosing period, or in the case of recovery group animals, at the end of the
treatment-free period, all animals were killed by intravenous overdose of sodium pentobarbitone
followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic
abnormalities were recorded

ORGAN WEIGHTS: (in order to determine general toxicological effects) Adrenals, Ovaries,Brain, Spleen,Epididymides, Testes, Heart, Thymus, Kidneys, Uterus and Liver

HISTOPATHOLOGY: (Yes) All tissues from control and 750 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesion was also processed.

Since there were indications of treatment-related liver and kidney changes, examination was subsequently extended to include similarly prepared sections of the liver and kidneys from all animals in the other treatment groups. The Mallory Heidenhain stain was also performed on kidney sections fromall males.

Microscopic examination was conducted by the Study Pathologist. All findings were entered into
the ROELEE Pathology computerisation system for tabulation and report production. Findings
were reported in the summary table by exception ( where a finding was seen it was reported but if
no finding was seen, the organ was not listed at all).
 
 
 
 
 
 
 
 
 
 
 

Other examinations:

No

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
All data were summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For
each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means
assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a
significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the
data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine
significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the
Mann-Whitney U test (non-parametric). Probability values (p) are presented as follows:
p < 0.01 **
p < 0.05 *
p > 0.05 (not significant)
Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes.
Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded
conditions. Probability values (p) are presented as follows:
p<0.001 +++ --- ***
p<0.01 ++ -- **
p<0.05 + - *
p<0.1 (+) (-) (*)
p>0.1 N.S. (not significant)
Plus signs indicate positive differences from the control group and minus signs indicate negative differences. Asterisks refer to overall between
group variation which is non-directional.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths and no clinically observable signs of toxicity were detected in test or control animals throughout the study period.
Increased salivation was evident in 750 mg/kg/day animals throughout the treatment period. Isolated incidents of increased salivation were also
evident in 200 and 50 mg/kg/day animals. Observations of this nature are often reported following oral administration of an unpalatable or slightly
irritant test material formulation and considered not to be an indication of systemic toxicity. Generalised red/brown stained fur and wet fur was
evident in a number of treated animals throughout the dosing period. Such observations are commonly observed in laboratory maintained rats and in view of the sporadic nature of these findings were considered to be entirely incidental and considered of no toxicological significance. One male
treated with 50 mg/kg/day had an open wound on day 28 followed by scab formation on days 29 to 31. One control male showed abnormal gait on
day 76. These were isolated incidental findings and unrelated to treatment.


BODY WEIGHT AND WEIGHT GAIN
No toxicologically significant effects on bodyweight development were detected.
Males treated with 750 mg/kg/day showed a statistically significant reduction in body weight gain during weeks 8 and 11 of treatment. Recovery 750 mg/kg/day males showed a statistically significant increase in body weight gain during week 16 (week 3 of the treatment free period). In the absence
of any effect on overall body weight gain, differences in weekly body weight change were considered to reflect normal biological variation and to be of no toxicological significance.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study) No feeding study
FOOD CONSUMPTION: There was no adverse effect on food consumption during the study period.


FOOD EFFICIENCY: Food efficiency (the ratio of body weight gain to dietary intake) was similar to that of controls.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study) No drinking water study

WATER CONSUMPTION: Daily visual inspection of water bottles revealed no intergroup differences.


OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related ocular effects.


HAEMATOLOGY
There were no toxicologically significant changes in the haematological parameters measured.
Non-recovery males from all treatment groups showed a statistically significant increase in erythrocyte count and a reduction in mean cell
haemoglobin. In the absence of any semblance of a dose related response the intergroup differences were considered to be of no toxicological
importance. Non-recovery females treated with 750 and 200 mg/kg/day showed a statistically significant reduction in activated partial
thromboplastin time. In the absence of an associated effect on clotting time the intergroup differences were considered to be of no toxicological
significance. Recovery 750 mg/kg/day males showed a statistically significant reduction in haemoglobin and haematocrit whilst recovery females
showed a statistically significant increase in platelet count following twenty eight days without treatment. In the absence of similar effects for
non-recovery animals at the end of the dosing period, the intergroup differences were considered to be incidental and ofno toxicological importance.


CLINICAL CHEMISTRY
Non-recovery males treated with 750 mg/kg/day showed statistically significant increases in plasma urea, calcium concentration, creatinine and
cholesterol with the majority of individual values for cholesterol and creatinine outside of the respective normal ranges for rats of the strain and age used. The effect on urea and creatinine extended to the 200 mg/kg/day non-recovery male dose group, with five male creatinine values being outside of the respective normal range (seeTable 1 A and B in " Remarks on results including tables and figures" and Table 4 in "Executive summary").
No such effects were detected in non-recovery females treated with 750 or 200 mg/kg/day, non-recovery animals of either sex treated with
50 mg/kg/day or recovery animals following twenty eight days without treatment (seeTable 1 A and B in " Remarks on results including tables and figures" and Table 4 in "Executive summary").
The remaining statistically significant intergroup differences were considered to be of no toxicological importance. Non-recovery females treated
with 750 and 200 mg/kg/day showed a statistically significant reduction in plasma glucose. The majority of individual values were within the
respective normal range and, in isolation, were regarded as incidental. Aspartate aminotransferase and alkaline phosphatase were reduced for
750 mg/kg/day non-recovery males. The majority of individual values were within the respective normal ranges and, in any case, only an increase in
these parameters are indicative of tissue destruction in the liver and kidneys and as such the reduction cannot be regarded as toxicologically
important. An increase in total protein was also detected in non-recovery females treated with 50 mg/kg/day. There was no dose related response
and as such the intergroup difference was considered to be fortuitous.


URINALYSIS
not examined


NEUROBEHAVIOUR
- Behavioural Assessments: there were no toxicologically significant changes in the behavioural parameters measured.
Hunched posture and tiptoe gait were evident in one male treated with 200 mg/kg/day during the open-field assessments performed in Week 9. In the absence of a dose related response or any similar effects being detected during the daily clinical observations this intergroup difference was
considered to be of no toxicological importance.
- Functional Performance Tests: there were no treatment-related changes in the functional performance parameters measured.
Females treated with 750 mg/kg/day showed a statistically significant increase in mean hind limb grip strength. This effect was confined to one out of the three tests for this parameter and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, this finding was
considered to be of no toxicological significance. Males treated with 200 mg/kg/day showed a statistically significant increase in overall activity. In
the absence of a dose-related response or any supporting clinical observations to suggest an effect of neurotoxicity, these findings were considered to be of no toxicological significance.
- Sensory Reactivity Assessments: there were no treatment-related changes in sensory reactivity.
All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used, and were of no toxicological importance.


ORGAN WEIGHTS
Males treated with 750 mg/kg/day showed a substantial increase in liver and kidney weight, both absolute and relative to terminal bodyweight,
compared with controls. Statistical significance was achieved and the majority of individual values were outside the respective normal ranges. The
effect on kidney weight extended to the 200 and 50 mg/kg/day male dose groups, again with many values outside the normally expected ranges.
Liver weights were also increased for 750 mg/kg/day females and in 200 mg/kg/day males (see Table 2 A in "Remarks on results including tables and figures" and Table 4 in "Executive summary").
The increased kidney weights persisted in recovery 750 mg/kg/day males following twenty eight days without treatment, however the intergroup
difference was not as substantial as in the non-recovery animals (see Table 2 B in "Remarks on results including tables and figures" and Table 4 in "Executive summary").
No toxicologically significant effects were detected in females treated with 200 or 50 mg/kg/day or in recovery 750 mg/kg/day females following a
twenty eight days treatment free period.
Non-recovery and recovery females treated with 750 mg/kg/day showed a statistically significant increase in kidney weight both absolute and relative to terminal bodyweight. In the absence of any histological correlates the intergroup differences were considered not to be of any toxicological
significance. Recovery 750 mg/kg/day females also showed a reduction in absolute and relative thymus weight. In the absence of similar effects for
non-recovery animals at the end of the dosing period, the intergroup differences were considered to be incidental and of no toxicological importance(see Table 2 A and B in "Remarks on results including tables and figures" and Table 4 in "Executive summary").

GROSS PATHOLOGY
Four non recovery males treated with 750 mg/kg/day showed enlarged kidneys at necropsy. One of the affected males also showed a speckled
appearance on the kidney surface and increased renal pelvic cavitation. Enlarged kidneys were also evident in one 200 mg/kg/day male.
The effect on the kidneys continued into recovery 750 mg/kg/day males following twenty eight days without treatment, with four males showing a
mottled appearance on the kidney surface(Table 3 -Summary Incidence of Necropsy Findings for non-recovery and recovery males (in "Attached background material") and Table 4 in "Executive summary")).
No such effects were detected in females treated with 750 or 200 mg/kg/day, in animals of either sex treated with 50 mg/kg/day or in recovery 750 mg/kg/day females following the treatment free period.


HISTOPATHOLOGY: NON-NEOPLASTIC
LIVER: Centrilobular hepatocyte enlargement was observed in relation to treatment for animals of either sex, but more especially for males, treated
with 750 mg/kg/day (P <0.001 for males and NS for females), and for males only treated with 200 mg/kg/day (P <0.001).
Three males treated with 50 mg/kg/day were also affected and the possibility that hepatocyte enlargement was related to treatment at this dose level cannot be excluded although such changes are occasionally seen among untreated rats as a spontaneous change.
Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated
inflammatory or degenerative changes, is generally considered to be adaptive in nature. This was evidenced by the regression of hepatocyte
enlargement among recovery 750 mg/kg/day animals following an additional twenty eight days without treatment (see Table 4 in "Executive summary").

KIDNEY: Tubular basophilia, tubular degeneration/necrosis and accumulations of globular eosinophilic material in the tubular epithelium were all
seen as a consequence of treatment for males only treated with 750 mg/kg/day (P <0.001 for all observations), or at 200 mg/kg/day (P <0.001 for
all observations). The majority of males treated with 50 mg/kg/day were affected only with accumulations of globular eosinophilic material in the
tubular epithelium (P <0.01).
Globular accumulations of eosinophilic material in the tubular epithelium are consistent with the presence of hydrocarbon nephropathy, which
results from the excessive accumulation of α2-microglobulin in renal proximal tubular epithelial cells. In more severe cases tubular basophilia and
tubular degeneration/necrosis are associated with α2-microglobulin accumulation. α2-Microglobulin is found only in the proximal tubular
epithelium of adult male rats.
Although globular accumulations of eosinophilic material had regressed to background control levels among recovery 750 mg/kg/day males, there was no evidence of regression of tubular basophilia or tubular degeneration/necrosis (P <0.001 for both conditions) following an additional
twenty eight days without treatment.
Further sections of kidney stained with Mallory Heidenhain were also examined. This staining technique demonstrates protein droplets in the renal
tubules and epithelium and correlated reasonably well with the eosinophilic accumulations reported from haematoxylin and eosin stained sections.
No adverse effects in male and female reproductive organs were detected.
All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were
no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance (Table 4 - Final summary of all adversed effects found in "Executive summary").

HISTOPATHOLOGY:
NEOPLASTIC: No effects

Dose descriptor:

NOAEL

Effect level:

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

Sex:

female

Basis for effect level:

other: gross pathology; organ weights; histopathology

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male

Basis for effect level:

other: gross pathology; organ weights; histopathology

Critical effects observed:

not specified

Data which only significant deviate or necessary for justification of the validity of this study's results are summarized in Tables 1 and 2:

Table1: Group mean blood chemical values for non-recovery animals

A. Males

Group (Sex)		Urea mg/dl	Glucose mg/dl	Tot. Prot. g/dl	Ca++ mmol/l	ASAT IU/l	AP IU/l	Creat mg/dl	Chol mg/dl
1(M)	Mean	22.6	149.1	7.568	2.532	72.9	279.3	0.790	76.2
	S.D.	4.9	17.4	0.530	0.047	8.0	77.2	0.067	7.5
2(M)	Mean	23.7	159.9	7.437	2.588	73.7	275.5	0.830	76.2
	S.D.	3.3	16.3	0.504	0.047	21.5	68.7	0.053	13.0
3(M)	Mean	26.6*	148.9	7.492	2.571	67.5	239.8	0.842*	79.6
	S.D.	4.6	13.7	0.440	0.079	4.7	41.5	0.063	15.5
4(M)	Mean	29.0**	152.0	7.473	2.607*	64.9*	189.2**	0.869**	89.6*
	S.D.	6.4	15.8	0.283	0.080	5.9	49.4	0.068	12.2

B. Females

Group (Sex)		Urea mg/dl	Glucose mg/dl	Tot. Prot. g/dl	Ca++ mmol/l	ASAT IU/l	AP IU/l	Creat mg/dl	Chol mg/dl
1(F)	Mean	29.7	146.1	7.015	2.611	73.9	183.7	0.846	77.2
	S.D.	6.3	20.3	0.365	0.093	11.5	67.0	0.071	14.1
2(F)	Mean	31.9	136.4	7.543*	2.685	81.1	188.8	0.903	82.2
	S.D.	6.2	10.2	0.257	0.053	15.6	73.2	0.106	21.6
3(F)	Mean	30.1	133.1*	6.972	2.619	78.3	184.6	0.828	82.3
	S.D.	5.8	7.4	0.357	0.102	11.3	77.4	0.070	16.7
4(F)	Mean	27.8	129.9**	7.130	2.583	78.0	159.7	0.813	87.4
	S.D.	4.1	8.8	0.569	0.104	13.5	52.3	0.046	19.7

Dose Levels: Group 1 - 0(Control) Group 2 - 50 mg/kg/day Group 3 - 200 mg/kg/day Group 4 - 750 mg/kg/day

M-males; F-females; 10 animals of each sex were used for every parameter tested. Asterixes indicate significance level.

Table 2: Group Mean with Corresponding Relative (% of Bodyweight) Organ Weights

A. Non-recovery animals

	MALES					FEMALES
		0 Control	50 mg/kg/day	200 mg/kg/day	750 mg/kg/day	0 Control	50 mg/kg/day	200 mg/kg/day	750 mg/kg/day
Kidneys	Mean (g)	3.15912	3.48128*	4.06481**	4.37218**	1.78743	1.68603	1.85559	1.81901*
	S.D.	0.39093	0.24750	0.65549	0.71916	0.17446	0.19862	0.19720	0.21460
	Mean (%)	0.543	0.594*	0.721**	0.785**	0.590	0.597	0.612	0.644*
	S.D.	0.046	0.050	0.095	0.114	0.056	0.039	0.053	0.039
Liver	Mean (g)	18.0617	19.3558	19.8321**	22.3936**	10.3658	9.95510	10.5018	10.9495*
	S.D.	1.87998	2.16781	2.51897	3.29017	1.18067	1.18859	0.92810	1.67206
	Mean (%)	3.107	3.289	3.508**	4.013**	3.420	3.531	3.463	3.884*
	S.D.	0.164	0.240	0.167	0.390	0.388	0.356	0.216	0.514
Thymus	Mean (g)	0.46297	0.46903	0.48859	0.46179	0.38588	0.36019	0.38674	0.35193
	S.D.	0.12032	0.07361	0.14691	0.08700	0.08699	0.07082	0.08189	0.10885
	Mean (%)	0.080	0.080	0.086	0.083	0.127	0.128	0.127	0.124
	S.D.	0.021	0.012	0.021	0.017	0.028	0.024	0.020	0.032

 

B. Recovery animals

		MALES		FEMALES
		0 Control	750 mg/kg/day	0 Control	750 mg/kg/day
Kidneys	Mean (g)	3.50237	3.97069**	1.71979	1.94648*
	S.D.	0.38627	0.43261	0.14790	0.12362
	Mean (%)	0.558	0.672**	0.585	0.608*
	S.D.	0.042	0.073	0.056	0.035
Liver	Mean (g)	19.3049	18.2855	9.99168	10.4391
	S.D.	1.89168	1.33113	1.04258	1.04399
	Mean (%)	3.077	3.093	3.397	3.261
	S.D.	0.139	0.208	0.370	0.335
Thymus	Mean (g)	0.45715	0.41172	0.34710	0.30426*
	S.D.	0.06759	0.07793	0.06566	0.06431
	Mean (%)	0.073	0.070	0.117	0.095*
	S.D.	0.009	0.013	0.018	0.019

M-males; F-females; 10 animals of each sex were used for every parameter tested. Asterixes indicate significance level

Conclusions:

The 'No observed adverse effect level' (NOAEL) for the purpose of hazard evaluation to human health is considered to be 200 mg/kg bw due to the specificity of effects in kidneys observed in male rats at low and mid dose level and to the fact that the effects demonstrated in the haematological and blood chemical parameter measured for 200 mg/kg/day in females were not considered toxicologically significant.

Executive summary:

The study was performed according abovementioned guidelines and was designed to investigate the systemic toxicity of the test material, by repeated oral administration to the Sprague-Dawley Crl:CD® (SD) IGS BR strain rat for a period of up to ninety consecutive days at dose levels of 50, 200 and 750 mg/kg/day. Recovery group of animals, which were maintained without treatment for a further twenty-eight days, was taken to determine reversibility or persistence of any toxic effects.

Clinical signs of toxicity, functional observations, bodyweight development, haematology, ophthalmoscopic examination, blood chemistry and food and water consumption as well histopathology were monitored during the study.

There was no apparent adverse effect on the physical condition of the animals, their water and food consumption.The statistically significant differences in weekly bodyweight change were considered to reflect normal biological variation and to be of no toxicological importance. There were no treatment-related ocular effects. The intergroup differences in the haemotological and the chemical blood parameters measured were considered to be incidental and of no toxicological importance.

Treatment-related effects in males at all dose levels and in females treated with 750 mg/kg/day were observed during the study. The increased kidney and liver weight in males, which persisted in recovery animals, correlated with histopathological findings in these organs. There was no similar effects in females. While elevated liver weight in males was considered to be a normal adaptive biological response to xenobiotics, an increase in kidney weight with the histopathological signs display "hydrocarbon nephropathy". This effect is not typical for females and other species and for this reason is not indicative of hazard to human health (see Table 4)

Table 4: Final summary of all advers effects found

Dose groups	Blood Chemistry			Increased organ weight						Gross pathology and histopathological endpoints
mg/kg bw				Kidney			Liver			Enlarged kidneys			Histopathology of liver			Histopathology of kidney
	M	F	Rec.	M	F	Rec.	M	F	Rec.	M	F	Rec.	M	F	Rec.	M	F	Rec.
50	-	-	-	+	-	-	-	-	-	-	-	-	+(slightly)	-	-	+(slightly)	-	-
200	+	-	-	+	-	-	+	-	-	+	-	-	+	-	-	+	-	-
750	+	-	-	+	+	-(f);+(m)	+	+	-(m,f)	+	-	-(f);+(m)	+	+(slightly)	-	+	-	-(m,f)

Following discussions with the Sponsor on how an argument could be made to suggest that all the effects detected in the kidneys were specifically associated with hydrocarbon nephropathy alone, which is a recognised phenomenon associated with the male rat only. Based on hazard evaluation the ‘No Observed Adverse Effect Level’ (NOAEL) should still be regarded as 50 mg/kg/day (due to tubular basophilia and tubular degeneration/necrosis at 200 mg/kg/day), however if the effects are all associated with hydrocarbon nephropathy then, based on risk assessment to humans, because hydrocarbon nephropathy is peculiar to the male rat, the NOAEL could be considered to be 200 mg/kg/day because of the specificity of the effect.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results. (Klimisch 1)

System:

other: Organ weight, gross pathological findings, histopathological findings (non-neoplastic), clinical biochemistry findings

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: 99.98% nitrogen

Details on inhalation exposure:

Exposure levels were determined three times daily by gas chromatography.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

6h/d and 5d/week for 13 weeks (at least 65 exposures)

Dose / conc.:

719 ppm (nominal)

Dose / conc.:

2 073 ppm (nominal)

Dose / conc.:

7 127 ppm (nominal)

Dose / conc.:

668 ppm (analytical)

Dose / conc.:

2 220 ppm (analytical)

Dose / conc.:

6 669 ppm (analytical)

No. of animals per sex per dose:

12 animals/sex/group

Control animals:

yes

yes, sham-exposed

Observations and examinations performed and frequency:

Exposure Chamber Monitoring (three times during exposure); Clinical Observations (twice daily); Body Weights and Food Consumption (All animals were weighed twice pretest, weekly during the study period, and prior to scheduled sacrifice. Food consumption was measured once during the week prior to treatment initiation and over a 6-d interval each week during the study period), Hematology and Clinical Chemistry (wk 14), Neurobehavioral Studies during wk 5, 9, 14 and 18 (recovery groups) (Motor Activity, Functional Operational Battery)

Sacrifice and pathology:

During week 14 (recovery groups (control and high dose only): week 18)

Statistics:

Statistical evaluations were performed on the following parameters: body weights, body weight change from wk 0, and food consumption; hematology and clinical chemistry; and organ weights, organ/terminal body weight ratio, and organ/brain weight ratio. Barlett's test at 1% significance, two-sided risk level, was used to determine if groups had equal variance. All other tests were conducted at 5% and 1% significance, two-sided risk level. Parametric procedures were standard one-way analysis of variance (ANOVA) using F distribution for significance. If significant differences among means were indicated, Dunnett's test was used to determine significant differences from controls. The Kruskal-Wallis test was the nonparametric procedure for testing equality of means, and if differences were indicated, Dunn's summea rank test was used to determine differences from controls.
A statistical test for trend in the dose levels was also performed, using standard regression techniques with a test for trend and lack of fit where variances were equal or Jonckheere's test for monotonie trend in nonparametric cases.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Increased incidence of red facial staining in both male and female rats

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Decrease in hemoglobin (5%), hematocrit (5%) and erythrocytes (7%) in blood of high dose males. These differences are within the historical range for control animals in this laboratory and therefore are not considered toxicologically relevant.

Urinalysis findings:

not examined

Dose descriptor:

NOEL

Effect level:

2 220 ppm (analytical)

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOEL

Effect level:

>= 6 646 ppm (analytical)

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

neuropathology

Dose descriptor:

LOEL

Effect level:

>= 6 646 ppm (analytical)

Sex:

male/female

Basis for effect level:

clinical signs

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Conclusions:

Systemic LOEL = 6646 ppm (24.3 g/m3) based on increased liver weight and red facial staining; NOEL = 2220 ppm (8.2 g/m3).
The Neurotoxicity NOEL = 6646 ppm (24.3 g/m3).

Executive summary:

Light alkylate naphtha (LAN, CAS #64741-66-8; approx 100% paraffinic) has been tested as a vapor distillate fraction (approx. 100% paraffinic) by inhalation in the rat for systemic toxicity and neurotoxicity, and in the rabbit by dermal exposure.

Sprague Dawley rats (12/sex/group) were exposed to a LAN light end distillate at concentrations of 0, 668, 2220, and 6646 ppm (2.44, 8.1 and 24.3 g/m3), 6 hours/day, 5 days/wk for 13 weeks, according to OECD guideline 413. The test material (LAN-D) was prepared to be representative of the fraction of light alkylate naphtha to which man would be exposed during normal handling and use. It was obtained by the distillation of light alkylate naphtha (LAN) and collection of that fraction that boiled over the temperature range 78 to 145°F. The maximum exposure level was 75% of the lower explosive limit for LAN distillate. Extra groups of 12 rats of each sex exposed to the high dose level and a recovery control group were maintained untreated for 28 days following cessation of the 13 weeks exposure. Neurobehavioral evaluations of motor activity and functional activity [FOB] were performed pretest and during weeks 5, 9, 14 and week 18 for recovery groups. Animals were not exposed to LAN-D during these tests. Ophthalmoscopic evaluations were performed pretest and just prior to the scheduled sacrifices at 14 weeks and 18 weeks (recovery groups). Body weights and food consumption were measured throughout the study. Blood samples were taken from 12 fasted rats/sex/group at 14 and 18 weeks for hematological and clinical chemical measurements. At termination (after 13 weeks exposure for the main study and after 18 weeks for the recovery animals) all animals were killed and subjected to a complete macroscopic examination. The following organs were weighed: adrenals, brain, heart, kidneys, liver, lung, ovaries, prostate, spleen, testes (with epididymides), thymus and uterus. Brain lengths and widths were measured for each rat. Thirty nine tissues removed from the control and high dose animals, were fixed, stained with hemotoxylin-eosin and examined histopathologically. Additionally, kidneys from selected animals were stained with Mallory-Heidenhain and examined. Tissues were collected from the nervous system (central and peripheral) of all animals and nervous system tissues were selected randomly from 6 rats per sex/group in the high dose and controls at the end of 13 weeks for microscopic examination. Specific brain regions examined were forebrain, cerebral cortex, hippocampus, basal ganglia, midbrain cerebellum and pons and medulla.

Neurobehavioral studies included motor activity, monitored as the number of beam breaks in an activity box, at pretest, and during weeks 5, 9, 14, and at the end of the 4- week recovery period. The Functional Operational Battery (FOB) was comprised of home cage evaluations, handling and open field behaviors and reflex assessment. Animals were also evaluated for fore limb and hind limb grip strength, landing foot splay and air righting ability.

There were no mortalities during the study and there were no treatment related signs of toxicity with the possible exception of an increased incidence of red facial staining in rats of both sexes in the high dose group. Mean body weights, body weight gains and food consumption were unaffected by treatment. Hematologic changes were a 5% decrease in hemoglobin and a 7% decrease in erythrocyte counts. Hemoglobin was still decreased 4% after the 4 week recovery period. However all these decreases were small and within historical control range for the laboratory. Decreases in AST and ALT in high dose females were not considered toxicologically significant because several control females had AST and ALT levels that were elevated relative to the other control females and relative to the historical control range. Comparison of values from high dose females with these elevated control values indicates that some were different by statistical criteria, but these differences were not toxicologically important. Organ weight changes were few. Statistically significant increases in kidney weights in high dose males correlated with microscopically observed hyaline droplet formation and degeneration of proximal renal tubules were observed, indicative of alpha 2-microglobulin mediated nephropathy, also identified as light hydrocarbon nephropathy, a species and sex specific syndrome not relevant to humans (US EPA, 1991). Increased liver weights in high dose rats of both sexes had no microscopic correlate and appeared reversible after 4 weeks of recovery. Absolute and relative liver weights were observed in the high dose males and females at 13 weeks but the differences had disappeared after the recovery period. There were no pathological findings associated with this increase. In the neurobehavioral studies no treatment-related effects were observed in the functional operational battery. In the study of motor activity there were some statistically significant differences, but overall they did not occur in a dose related manner and furthermore were smaller than some of the differences seen during the pre-dosing period. The systemic LOAEL = 6646 ppm (24.3 g/m3) based on increased liver weight and red facial staining and the NOAEL = 2220ppm (8.2 g/m3). The Neurotoxicity NOAEL = 6646 ppm (24.3 g/m3).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

8 200 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Study well documented, meets generally accepted scientific principles, acceptable for assessment and GLP compliant (Klimisch 2).

System:

other: increased liver weight

Organ:

liver

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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

Repeated dose toxicity: oral

Ninety day repeated dose oral (gavage) toxicity study in the rat was performed to investigate systemic toxicity of C4 -10 branched and linear hydrocarbons. Test material was applied by gavage to rats at dose levels of 50, 200 and 750 mg/kg bw. Control group was dosed with vehicle alone (Arachis oil BP). Two recovery groups were treated with the high dose (750 mg/kg/day) or the vehicle alone for ninety consecutive days and then maintained without treatment for a further twenty-eight days. According OECD guideline 408 a full set of observation and examinations was performed in this study.

There were no unscheduled deaths in this study. No toxicologically significant effects were identified in clinical observations, behavioural parameters, functional performance tests, or sensory reactivity assessments. Bodyweight and bodyweight gain, food and water consumption, ophthalmoscopic evaluation and haematology parameters were not adversely affected by treatment with the test material.

Treatment related findings were observed in male rats at high (750 mg/kg bw/day) and mid (200 mg/kg bw/day) dose levels. Blood chemistry parameters as well as liver and kidney weights were statistically significant increased at these dose levels. In females, some effects were demonstrated in the haematological and blood chemical parameter measured for 200 mg/kg/day dose group. At necropsy, male animals showed enlarged kidneys at high dose level and partially at mid dose level. Except increased liver weight at 750 mg/kg bw, no such effects were detected in female animals. At histopathology, centrilobular hepatocyte enlargement was displayed in males treated with 750 and 200 mg/kg bw to a greater extent than in females. This effect, however, was considered to be adaptive in nature. Regarding observed effects in kidneys, only males treated with 750 and 200 mg/kg bw were affected with tubular basophilia, tubular degeneration/necrosis and accumulations of globular eosinophilic material in the tubular epithelium. The majority of males treated with 50 mg/kg/day were affected only with accumulations of globular eosinophilic material in the tubular epithelium.

According to the study report, the kidney changes identified in males at all doses (globular accumulations of eosinophilic material) are consistent with well documented light hydrocarbon-induced nephropathy peculiar to male rats and are not considered indicative of a hazard to human health by the US EPA or EU regulatory agencies. Excluding the effects demonstrated in the haematological and blood chemical parameter measured for 200 mg/kg/day females, which were not considered toxicologically significant, the NOAEL relevant for risk assessment to humans is established to be 200 mg/kg/day for both sexes.

Repeated dose toxicity: inhalation

A closely related substance with limited range, Light alkylate naphtha (LAN, CAS #64741-66-8; approx. 100% paraffinic), has been tested as a vapor distillate fraction (approx. 100% paraffinic) by inhalation in the rat for systemic toxicity and neurotoxicity, and in the rabbit by dermal exposure.

Sprague Dawley rats (12 group/sex) were exposed to a LAN light end distillate at concentrations of 0, 668, 2220, and 6646 ppm (2438, 8102 and 24300 mg/m3), 6 hours/day, 5 days/wk for 13 weeks, according to OECD guideline 413. The test material (LAN-D) was prepared to be representative of the fraction of light alkylate naphtha to which man would be exposed during normal handling and use. It was obtained by the distillation of light alkylate naphtha (LAN) and collection of that fraction that boiled over the temperature range 78 to 145°F. The maximum exposure level was 75% of the lower explosive limit for LAN distillate. Extra groups of 12 rats of each sex exposed to the high dose level and a recovery control group were maintained untreated for 28 days following cessation of the 13 weeks exposure. Neurobehavioral evaluations of motor activity and functional activity (FOB) were performed pretest and during weeks 5, 9, 14 and week 18 for recovery groups. Animals were not exposed to LAN-D during these tests. Ophthalmoscopic evaluations were performed pretest and just prior to the scheduled sacrifices at 14 weeks and18 weeks (recovery groups). Body weights and food consumption were measured throughout the study. Blood samples were taken from 12 fasted rats/sex/group at 14 and 18 weeks for hematological and clinical chemical measurements. At termination (after 13 weeks exposure for the main study and after 18 weeks for the recovery animals) all animals were killed and subjected to a complete macroscopic examination. The following organs were weighed: adrenals, brain, heart, kidneys, liver, lung, ovaries, prostate, spleen, testes (with epididymides), thymus and uterus. Brain lengths and widths were measured for each rat. Thirty nine tissues removed from the control and high dose animals, were fixed, stained with hemotoxylin-eosin and examined histopathologically. Additionally, kidneys from selected animals were stained with Mallory-Heidenhain and examined. Tissues were collected from the nervous system (central and peripheral) of all animals and nervous system tissues were selected randomly from 6 rats per sex/group in the high dose and controls at the end of 13 weeks for microscopic examination. Specific brain regions examined were forebrain, cerebral cortex, hippocampus, basal ganglia, midbrain cerebellum and pons and medulla.

Neurobehavioral studies included motor activity, monitored as the number of beam breaks in an activity box, at pretest, and during weeks 5, 9, 14, and at the end of the 4- week recovery period. The FOB was comprised of home cage evaluations, handling and open field behaviors and reflex assessment. Animals were also evaluated for fore limb and hind limb grip strength, landing foot splay and air righting ability.

There were no mortalities during the study and there were no treatment related signs of toxicity with the possible exception of an increased incidence of red facial staining in rats of both sexes in the high dose group. Mean body weights, body weight gains and food consumption were unaffected by treatment. Hematologic changes were a 5% decrease in hemoglobin and a 7% decrease in erythrocyte counts. Hemoglobin was still decreased 4% after the 4 week recovery period. However all these decreases were small and within historical control range for the laboratory. Decreases in AST and ALT in high dose females were not considered toxicologically significant because several control females had AST and ALT levels that were elevated relative to the other control females and relative to the historical control range. Comparison of values from high dose females with these elevated control values indicates that some were different by statistical criteria, but these differences were not toxicologically important. Organ weight changes were few. Statistically significant increases in kidney weights in high dose males correlated with microscopically observed hyaline droplet formation and degeneration of proximal renal tubules were observed, indicative of alpha 2-microglobulin mediated nephropathy, also identified as light hydrocarbon nephropathy, a species and sex specific syndrome not relevant to humans (US EPA, 1991). Increased liver weights in high dose rats of both sexes had no microscopic correlate and appeared reversible after 4 weeks of recovery. Absolute and relative liver weights were observed in the high dose males and females at 13 weeks but the differences had disappeared after the recovery period. There were no pathological findings associated with this increase. In the neurobehavioral studies no treatment-related effects were observed in the functional operational battery. In the study of motor activity there were some statistically significant differences, but overall they did not occur in a dose related manner and furthermore were smaller than some of the differences seen during the pre-dosing period. The systemic LOAEL = 6646 ppm (24300 mg/m3) based on increased liver weight and red facial staining and the NOAEL = 2220 ppm (8200 mg/m3). The Neurotoxicity NOAEL = 6646 ppm (24300 mg/m3).

In a supporting study, male and female rats were treated by inhalation with the same test material, Light alkylate naphtha (petroleum) at concentrations of 1.54, 4.92 and 15.31 mg/L during 21 days. No systemic effects were found in female animals, but male animals displayed light hydrocarbon nephropathy. There was a 100% incidence of tubular dilation and necrosis at the corticomedullary junction of the kidneys in male rats for all three exposure groups.

The results of this study are consistent with those of the repeated dose toxicity oral study and confirm that hydrocarbon nephropathy is peculiar only to the male rat in response to treatment with some hydrocarbons and for this reason, the effect is not indicative of hazard to human health.

Justification for classification or non-classification

Based on available data of 'Naphtha (Fischer-Tropsch), light, C4 -10 branched and linear' and closely related substances, classification is not warranted according to the criteria of EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

The self classification STOT Repeated Exp. 2 (H373) is based on the classification of the constituent n-hexane (presence of n-hexane in the test material up to 20%).