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Description of key information

The dermal and oral toxicity of AHTN are determined by a number of studies. Based on the lowest reliable LD50 for AHTN, the classification of category 4 of the acute oral toxicity applies (Harmful if swallowed).

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 August 1985 - 23 August 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with GLP requirements and OECD 401 method.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Deviations:
yes
Remarks:
The variation in volume of dosing; that recommendation was not part of that guideline.
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
other: Cpb:Wu;Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Young adult, SPF—bred albino rats (Cpb:Wu;Wistar random) were used. They were obtained from the Central Institute for the Breeding of Laboratory Animals TNO, Zeist, the Netherlands. The body weights of the males varied from 245 to 283 g, those of the females from 162 to 187 g. The rats were kept under the environmental conditions of the Institute’s animal house for 1 week prior to the test.
Species: albino ratis, male: weight 245-283 g and female: weight 162-187 g.Test conditions: temperature: 22 =/-2 degreesCelsius. Relative humidity; 40-60 % 12 hours light/12 hours dark. Food: cereal-based diet.
Route of administration:
oral: gavage
Vehicle:
other: isopropyl myristate (IPM)
Doses:
17.5% Tonalid in IPM. Dose: 4.00, 4.80, 5.76, 6.91, 8.29 ml/kg body weight euivalent to 700, 840, 1008, 1209, 1451 g/kg body weight.
No. of animals per sex per dose:
10 (5 male and 5 female)
Control animals:
no
Statistics:
The LD5O was calculated according to the method of Weil, C.W., Biometrics 8 (1952) 249-263
Sex:
male/female
Dose descriptor:
LD50
Effect level:
920 mg/kg bw
95% CL:
> 795 - < 1 066
Mortality:
Deaths occurred between 6 hours and 8 days after dosing.
Clinical signs:
Within a few hours after dosing the rats showed sluggishness and piloerection. - Later on haematuria was observed until 2 days after treatment. After a few days, moreover, encrustations around eyes and nostrils, and signs of’ emaciation were observed. These latter phenomena persisted in all surviving rats during the first post—treatment week.
In the second post—treatment week the survivors recovered gradually and looked quite healthy again at the end of the observation period.
Body weight:
The individual body weights on day 7 revealed growth retardation or weight loss of all survivors. On day 14 the body weight of most of the rats was normal again for rats of this strain and age.
Gross pathology:
Macroscopic examination at autopsy of the rats that died in the first few post—treatment days revealed blood—stained urine in the bladder of all rats. In the rats that died thereafter and in those that survived the observation period no treatment—related gross alterations were found.

Table 1 Body weight and mortality rate in rats after a single oral dose administration in rats

Bodyweights on day (g) Mortality
Dose Males Females Males Females
mg/kg bw 0 days 7 days 14 days 0 days 7 days 14 days
700 268 280 315 172 171 191 1/5 0/5
840 254 212 273 170 157 170 4/5 0/5
1008 266 235 287 178 150 196 3/5 4/5
1209 257 250 304 174 152 175 4/5 3/5
1451 260 - - 173 - - 5/5 5/5
Interpretation of results:
Category 4 based on GHS criteria
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
After treatment, the rats showed sluggishness, piloerection, haematuria, encrustations around eyes and nostrils & signs of emaciation. Deaths occurred between 6 hours and 8 days after treatment. The oral LD50 of Tonalid was found to be 920 mg per kg body weight with 795 and 1066 as the 95% confidence limits.
Executive summary:

Groups of five male and five female young adult albino rats were administered by gavage 4.00, 4.80, 5.76, 6.91 or 8.29 ml/kg bw of a 17.5% (w/v) solution of AHTN (Tonalid®; purity =98%; equivalent to doses of 700, 840, 1008, 1209 and 1451 mg/kg bw AHTN) in isopropyl myristate and observed for 14 days. Within a few hours, signs of sluggishness and piloerection were seen. Later on haematuria, encrustrations around eyes and nostrils and signs of emaciation were observed. Death occurred between 6 hours and 8 days of dosing at which time survivors recovered gradually looking healthy at the end of the observation period. Macroscopic examination at autopsy of the rats that died in the first few days revealed blood stained urine in the bladder. No other treatment-related gross alterations were seen in these or other animals. An LD50 of 920 mg/kg bw was calculated.

 

Source: EU Risk Asssessment Report AHTN, Luxembourg, Office for Official Publications of the European Communities, ECB (May 2008)

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
920 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 June 1977 - 12 August 1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was conducted prior to GLP and OECD guidelines, but was according to acceptable procedures at that time.
Principles of method if other than guideline:
Sprague-Dawley rats were administered AHTN in absolute ethanol (or undiluted) by dermal application to the shaved skin, and were observed for mortality and clinical effects immediately after the dosing at 1, 4, and 24 hours, and also once daily thereafter for a total of 7 days.
GLP compliance:
not specified
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
Initial body weight ranged from 108 to 197 grams, and the animals were fasted overnight prior to dosing.
Type of coverage:
not specified
Vehicle:
ethanol
Details on dermal exposure:
AHTN was administered by gentle inunction to the shaved skin of 5 groups of five female rats
Duration of exposure:
single administration of dose
Doses:
0.5 to 10 g/kg
No. of animals per sex per dose:
5 (5 groups of 5 animals)
Control animals:
no
Statistics:
Mortality data was analyzed statistically, utilising the tables of Horn, H.J., Biometrics 12, 311, 1956.
Sex:
female
Dose descriptor:
LD50
Effect level:
7 940 mg/kg bw
95% CL:
4 890 - 12 900
Remarks on result:
other: Assuming all dead at 21500 mg/kg.

Major necropsy findings: animals found dead exhibited the effects expected at their respecive stages of autolysis; all other animals exhibited no remarkable necroscopy findings.

Table 1: Mortality data and toxic effects of Fixolide tested on acute dermal toxicity on rats.

Dose Concentration  Time of death
g/kg bw % in ethanol Immediate 1-24 h. 24-48h.
0.464 10 0/5 0/5 0/5
1.000 10 0/5 0/5 1/5
2.150 10 0/5 0/5 0/5
4.640 20 0/5 0/5 1/5
10.000 40 0/5 0/5 3/5
Interpretation of results:
GHS criteria not met
Conclusions:
LD 50 AHTN = 7940 mg/kg, which is larger than the upper limit for classification dermal toxicity (i.e. 2000 mg/kg).
Executive summary:

Groups of 5 female rats were fasted overnight prior to the experiment, and the test material in absolute ethanol was applied to the shaved skin of the animals. Observations for mortality and clinical effects were conducted immediately after the dosing at 1, 4, and 24 hours, and also once daily thereafter for a total of 7 days. Gross necropsy was conducted on all animals (those that died or were sacrificed at the end of the study). The LD50 value was 7.94 g/kg. Animals found dead exhibited the effects expected at their respective stages of autolysis; all other animals exhibited no remarkable necropsy findings.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
7 940 mg/kg bw

Additional information

AHTN should be classified as "Harmful if swallowed" according to Table 3.1.2 of the CLP regulation (EC 1272/2008). The EU Risk Assessment report mentioned LD50 of an oral intubation study from Minner [1] of 825 mg/kg, while an intraperitoneal study in the same report showed the lowest LD50 of 79 mg/kg. Nevertheless, the latter study is very old, not conducted according to an OECD method and not conducted by a GLP accredited company.

An intraperitoneal GLP study on mice was performed by Gudi [2] showing a LD50 of ca. 2000 mg/kg. Three tests from Spanjers (1985) showed similar LD50 values and all three concluded that AHTN should be classified as harmful if swallowed. The lowest LD50 (oral) obtained from the key studies was selected to carry forward in the risk assessment (Spanjers 1985a).

The three oral toxicity studies from Spanjers (1985) show steep dose response curves (see attachment "figure acute toxicity mortality rats"). The key study used in determining the LC50 shows a LOAEL of around 700 mg/kg bw. The mortality rate in the Spanjers 1985b curve starts already at 40% for the lowest dose in that study. The curves in the study Spanjers 1985b and 1985c have shifted towards higher concentrations. The mortality rate in the Spanjers 1985b study is already 40% at the lowest concentration. The study of Spanjers 1985c confirms the study of Spanjers 1985a even though it contains one outlier in the dose-response curve.

Loss in weight has been described extensively in several studies, also in other toxicity studies. However, this weight loss is fully reversible after the exposure period and although this is a sub-lethal adverse effect, only the acute toxicity for a single exposure is considered here.

AHTN should not be used for human ingestion and exposure is purely of accidental nature.

Based on the three studies, taking the LOAEL of the most conservative curve (Spanjers 1985a), the LOAEL can be converted into a NOAEL by applying an assessment factor of 3.

Referenced in EU Risk Assessment Report AHTN, ECB (May 2008)

[1] Minner and Foster, Comparative acute toxicity studies in the female rat with five synthetic musk chemicals, 1977

Justification for classification or non-classification

The test substance has to be classifiedas category 4 for acute oral toxicity according to Regulation (EC) No 1272/2008, based on the acute oral toxicity test (Spanjers 1985a).